Microbiology Tests

The timely detection and/or laboratory diagnosis of infectious diseases is critical to the prevention of death due to most infectious diseases in most parts of the world especially in developing economies such as in sub-Saharan Africa, parts of Asia and South America. It is vital to accurately detect invading disease causing agents in the face of any infection, and this should be timely done so that therapy can be properly guided. This chapter updates the reader on the various techniques used to analyze clinical specimens as is obtainable in the microbiology laboratory. Microbiology unlike other biological sciences is a fascinating and rewarding discipline that revolves around life at the minute state (i.e. microorganisms). There is actually no microbiology without laboratory modus operandi. Microbiology like all the other biological disciplines is founded on different laboratory and/or experimental procedures that clarify the working nature of microorganisms, their synthesized products and their activity in the environment.

The microbiology laboratory deals mainly with the isolation, identification and/or characterization of microorganisms amongst other procedures from virtually all materials emanating from humans or the environment in a view to harnessing their beneficial importance as well as coming up with ways of halting their untoward effects to man and his immediate environment. There are numerous hazards that can be encountered in the microbiology laboratory if students and teachers alike including scientists fail to work according to laid down laboratory guidelines when carrying out their research works. As explained earlier in this textbook, students and all users of the microbiology laboratory are advised to adhere strictly to all the rules and regulations of the laboratory as well as ensure that aseptic technique is imbibed always to ensure better results from their laboratory investigations and ultimately to avoid been infected with the infectious organisms. Microorganisms are ubiquitous (i.e. they are everywhere); and this includes both the beneficial and harmful microbes. Thus, it is critical for all users of the microbiology laboratory to follow the laboratory precautions that are customary to their own laboratory so as to work safely and protect the lives of others and even their immediate working environment.

In the ensuing pages of this textbook, the fresher students in microbiology including those who already have mastery of the subject shall be taken on an expedition of exploring some of the basic laboratory protocols that makes up the microbiology laboratory. This chapter covers bacteriological investigations, investigations in parasitology, serology and mycology; and the analysis of a wide variety of clinical specimens including but not limited to sputum, blood, feaces and urine shall be expanded. The following foundational laboratory techniques in bacteriology, mycology, serology, and parasitology shall sustain microbiology students in their quest for higher knowledge in the microbial world of life. Students learn more when they are involved in a particular process rather being told or shown how to do things.

This section of this textbook has been designed to involve students in virtually all the foundational laboratory techniques that are exclusive to the microbiology laboratory. It is expected that all students of microbiology shall demonstrate optimum mastery of microbiology after going through the following pages in a thoughtful and reflective manner. Readers are advised to know that the reliability and reproducibility of a test result can be affected by some factors which could eventually lead to some errors in the reliability of the final test result. Some of these factors which could be controlled include the type of instruments/equipments used, laboratory personnel’s’ experience, testing reagents and methods, environmental factors and the quality of samples collected for the analysis. However, this chapter has been prepared to give a concise description of some of the laboratory techniques attempted in the microbiology laboratory.

BACTERIOLOGY

Bacteriology is simply defined as the scientific study of bacteria. For the purpose of this book, only pathogenic bacteria (i.e. those bacteria that cause diseases and infections in humans) shall be the prime focus of this chapter. Pathogenic bacteriology thus, is the scientific study of bacteria that cause diseases in man. This unit which is one of the most important units of the clinical microbiology laboratory handles different types of patient’s specimens, with the sole aim of growing and isolating pathogenic bacteria from clinical specimens so that treatment can be properly administered by the physician. The proper and adequate isolation and growth of bacteria for which this unit is known for, are required before any biochemical tests can be used to confirm the identification of the pathogen isolated. It was said in the preceding pages of this work that the sole aim of the clinical microbiologist is to isolate and grow pathogens from host tissue and specimens so as to define the cause of an infectious disease which has taken hold of a patient. This is indeed true as it will be expanded in this chapter how the clinical microbiologist goes about achieving this prime aim.

The direct physical and microscopic examinations of the smears of patient’s specimens and other environmental samples cannot be sufficient enough to characterize and identify specifically the different species of microorganisms present in the sample. Thus it is paramount to undertake specific identification techniques in identifying the species of bacteria in the specimen using cultural technique. The presence of bacterial growth in the microbiology laboratory can be usually recognized by the development of colonies on solid media or turbidity/cloudiness in liquid media (broth). Cultivation is defined as the process of propagating/growing organisms (e.g. bacteria) in the laboratory by providing them with the proper and adequate environmental conditions needed for their growth. In a more general term, this process also means culture/culturing of microorganisms.

To grow or culture a microorganism simply means to make replicas of the microorganism, and doing this requires the microbiologist to provide all the needed nutrients with which the growth of the organism can be propagated. The technique of doing this in the laboratory is called culture technique, and this is one of the most significant techniques that differentiate the clinical microbiology laboratory from other laboratories like hematology, clinical chemistry e.t.c in a hospital setting. It is a major technique in the clinical microbiology laboratory by which the causative agent of a particular disease can be defined. The culture of a microorganism is carried out in a solid or liquid medium which is totally made sterile (i.e. free from all microorganism). The culture media plate is then inoculated with a specimen or an organism and then incubated at a specific temperature and time that supports growth of the microorganism. A pure culture (which is a culture that contains only a single kind of organism) which is usually free from any form of contaminating microorganism is finally recovered from the culture media plate after incubation. The isolated pure culture is then further tested biochemically and molecularly in order to identify and characterize them.

Apart from demonstrating the presence of a pathogen which may be causing a particular disease, the cultural technique in microbiology also helps to provide a platform via which microorganisms can be studied in a more accurate way based on their different growth patterns on culture media. It also enables colonies of pure bacterial growth to be isolated for further identification, biochemical, immunological, and antimicrobial susceptibility testing’s. Microorganisms vary widely in their nutritional demands/requirements and their sources of metabolic energy. Thus there are also a wide variety of media (microorganism’s food) available for the proper isolation of bacteria depending on the type of bacteria to be isolated. Enjoy yourself!

PREPARATION OF BACTERIAL SMEAR

 Bacterial smear is defined as a dehydrated or dried preparation of a bacterial suspension (cells) on a clean glass slide. The source of bacteria for the preparation or making of a slide can be from an agar slant, broth (liquid) culture and from a culture plate.

Procedure of making a bacterial smear

  • Using a grease pencil, mark one end of the slide with the name of the bacterial culture.
  • Transfer a speck of the bacteria to a drop of normal saline on a clean glass slide. If the bacteria is from a liquid medium (i.e. broth), place 1-2 drops or loops of bacteria on a clean glass slide.
  • Spread out the bacteria (from a broth or solid culture) mixture over a large area on the glass slide. Always ensure that a thin preparation is made and, that a space is left on each of the four sides of the glass slide.
  • Air dry the glass slide at room temperature (25-28oc) while ensuring that it is free from dust.
  • Heat-fix the smear by passing it through a Bunsen burner flame three times or by flooding the smear with 70% methanol or ethanol and allowed for 2 min. Always ensure that the prepared smear is on top of the glass slide as you pass it through the Bunsen burner flame.
  • After heat-fixing, the fixed smear can be stained on a staining rack using different types of dyes depending on the staining technique used. There are different staining techniques that are available in the bacteriology laboratory. The staining can be simple or Some of the staining techniques used in the bacteriology laboratory include:
  • Gram staining: For dividing bacteria into either Gram positive or Gram negative.
  • Ziehl-Neelsen staining: For detecting acid-fast bacilli e.g. Mycobacterium.
  • Albert staining: For detecting Corynebacterium diphtheria from sputum specimen.
  • Wayson staining: For detecting Yersinia pestis
  • Loeffler methylene blue stain: For detection of Bacillus anthracis.
  • Malachite green staining: For detection of bacterial endospores.
  • Crystal violet staining: For detection of bacterial spores.
  • Robinow’s staining: For detection of bacterial DNA.
  • Capsule staining: For detection of bacterial capsules.
  • Add a drop of immersion oil on the stained slide.
  • Examine the slide under the oil immersion objective lens (x100)

Heat-fixing is defined as the microbiological technique of attaching bacterial cells onto the surface of a glass slide by the use of a Bunsen burner flame or 70% of methanol. A gentle heating of a bacterial cell on a slide when passed through the blue flame of a Bunsen burner actually kills the bacteria but nevertheless, heat-fixing does not distort the cell structure of the organism. The cell structure of the bacteria cell becomes distorted if and only when the slide is over-heated. Heat-fixing of bacterial cells allows the internal and external structures of a microorganism to be intact during staining and microscopy.

Techniques of making good bacterial smear

  • Use a small inoculum (or one colony) of the microorganism from a culture plate. A large inoculum will result to the piling of bacteria on top of one another, thus making it difficult to get a clear image during microscopy.
  • One or two loops of the bacterial cell suspension should be placed on a clean glass slide and spread out over a large area. This technique applies to a liquid medium containing a bacterial cell.
  • Inoculum of bacterial cell from a culture plate should be diluted with one or two drops of physiological (normal) saline and spread out properly.
  • Allow the prepared slide to dry properly before staining and subsequent microscopy.
  • Glass slides containing smears of bacteria should be kept free from dust particles.

Qualities of a well prepared bacterial smear 

  • The shape of the bacterial cell will not be distorted or unclear during microscopy.
  • The bacteria will be properly and evenly separated from one another after spreading out on the slide and subsequent microscopy.
  • During staining of the prepared smear, the bacteria will not be washed off the slide

 GRAM STAINING

Gram staining is a general purpose bacteriological identification technique used in the bacteriology section of the microbiology laboratory to identify and differentiate bacteria into two groups i.e. Gram-positive and Gram-negative. It was discovered by Christian Gram (18531938), a Danish scientist in 1884. Gram showed that the cells of some bacteria could be easily decolorized with organic solvents (e.g. ethanol) after staining with some basic dyes (e.g. crystal violet) while the cells of other group of bacteria (known as Gram-positive bacteria) resisted the decolorization after the staining process. Gram staining technique is generally used to identify bacterial pathogens from cultures and clinically important specimens based on their Gram reaction.

Bacteria that are neither Gram-positive nor Gram-negative are termed Gram-variable organisms and such bacteria cannot be identified with the Gram staining technique. Gram variable bacteria are usually old bacterial cultures that have lost their cell wall structure over time. Gram indeterminate bacteria are those bacteria that cannot be detected by Gram staining technique. Typical example of Gram indeterminate bacterium is Mycobacterium species that posses a waxy/lipid-containing cell wall (that contains mycolic acid). In the Gram staining technique, Gram-positive bacteria stain blue-violet while Gram-negative bacteria stain red.

Gram staining is one of the most important differential staining techniques used by microbiologists worldwide to characterize bacteria based on their cell wall compositions. Crystal violet (primary stain), Lugol’s iodine (mordant), ethanol/acetone (decolorizer) and safranin (secondary or counter stain) are the stains or dyes used for carrying out Gram staining technique in the laboratory. Gram-positive bacteria retain the primary stain after decolorization while Gram-negative bacteria do not. The mordant (iodine) increases the interaction of the bacterial cell wall with the primary stain while the decolorizer removes the primary stain from the cell wall of the test bacteria (i.e. if the organism is Gram-negative). Gram-negative bacteria retain the colour of the counter stain, and appear red under the microscope.    

 

Illustration of Gram positive stain result. The stained organism is Gram-positive cocci.

 

Illustration of Gram negative stain result. The stained organism is Gram-negative bacilli

PROTOCOL FOR GRAM STAINING TECHNIQUE

  • Prepare a thin smear of the test bacteria on a clean glass slide. Smear preparation is described in detail in the top part of this section.
  • Allow the prepared smear to dry while standing on a staining rack.
  • Add crystal violet stain using a dropper. Allow for 60 seconds.
  • Wash off the stain with clean water.
  • Add or cover the smear with Lugol’s iodine using a dropper. Allow for 60 seconds.
  • Wash off the stain with clean water.
  • Add a drop of acetone or ethanol to decolorize the stained smear.
  • Wash off with water immediately.
  • Add safranin to the stained smear. Allow for 60 seconds or a maximum of 2 minutes.
  • Wash off the stain with clean water. Allow to dry while standing on the staining rack. Ensure to carefully clean the back of the stained slide with a clean cloth or cotton wool.
  • Add a drop of immersion oil on the stained smear.
  • View the stained smear under the microscope using the oil immersion objective lens (100X).
  • Gram-positive bacteria appear blue-violet under the microscope while Gram-negative bacteria appear red. Ensure to take notice of the bacteria shape (i.e. cocci, rod, coccobacilli or streptococci) when reporting the result of a Gram stained smear.

STOOL CULTURE

Stool culture is demanded in the bacteriology laboratory as method for detecting and diagnosing enteric bacterial infections (i.e. infections caused by pathogens in the Enterobacteriaceae family e.g. Salmonella species and Shigella species) that lead to enteric fever, diarrhea and dysentery.

AIM: To isolate and identify pathogenic bacteria from feacal specimen culture as an aid in the diagnosis of gastrointestinal infections.

MATERIAL/APPARATUS:  Stool specimen, salmonella shigella agar (SSA), MacConkey agar (MCA), Bunsen burner, inoculating loop, incubator, grease pencil.

SSA = Salmonella-Shigella agar

MCA = MacConkey agar

METHOD/PROCEDURE:

  1. Label the SSA and MCA plates using a grease pencil.
  2. Using a sterilized inoculating loop, collect a speck or loopful of the stool specimen.
  3. Inoculate it on the SSA and MCA plates respectively. Note: The inoculating loop should be flamed or sterilized intermittently as the streaking continues.
  4. Incubate both plates in the incubator at 37oC overnight.
  5. After incubation, bring out the plates and look for colonies of Shigella or Salmonella on the SSA plate, and colonies of Escherichia coli and other lactose fermenting organisms on the MCA plate.
  6. Subculture suspect colonies of bacteria to a new SSA and MCA plates in order to obtain a pure culture – which is the basis for any meaningful identification of a bacteria. The above procedure should be followed aseptically. But this time, the stool specimen is not used but the isolated organism in both the SSA and MCA plates.
  7. Perform biochemical testing on the isolated pure culture organisms in order to identify the isolate organisms. The biochemical tests to perform are: urease test, indole test, citrate test, and sugar test using triple sugar iron agar.
  8. Perform antimicrobial susceptibility test only when a pathogen is successfully isolated and identified.

NOTE: If the stool specimen is very thick that obtaining a loopful of it will be difficult, make a thick suspension of it in about 1ml of sterile peptone water. Take a loopful from the stool specimen in the peptone water tube and perform your inoculation.

SSA plate is for the isolation of Salmonella and Shigella. Both are non – lactose fermenters (NLF), and appear as a pale or colourless colonies on the SSA plate. Xylose – lysine deoxycholate (XLD) agar can also be used in place of SSA as both perform almost the same function.

MCA PLATE is for the isolation of lactose fermenters (LF) like E. coli. E. coli and other lactose fermenting organisms appear as pinkish colonies on the MCA plate.

REPORTING OF THE RESULT:

Examine the plates and report the cultures. The culture of a stool specimen, and subsequent isolation and identification of any pathogen present may take about 3 – 4 days before any final conclusions can be reached. Depending on whether any meaningful isolation and identification was made or not, stool culture results can be reported in any of the following ways:

  • Cultures yield no significant growth.
  • Cultures yield no bacteria growth.
  • Cultures yield Enteropathogenic coli. Before you can confidently say that cultures yield a particular organism, you must have carried out all the necessary biochemical tests and serotyping required for identifying a pathogenic bacterium. It is advisable to stick to and operate by the standard operating procedures (SOP’s) of the laboratory in which you are doing the work, as the SOP’s for laboratories vary. Also, familiarization with the different types of bacteria implicated in causing intestinal infections and how they can be identified and differentiated from each other is an advantage.

BLOOD CULTURE

Blood culture is the most important diagnostic method for detecting and diagnosing bacteraemia and fungimia in the clinical microbiology laboratory. Blood specimen required for blood culture technique should be collected from the patient prior to antibiotic therapy in order to increase the sensitivity of the test. This is important because prior antibiotic therapy before the drawing of blood for blood culture reduces the effectiveness of the test. It is therefore a general rule to collect blood samples for blood culture techniques before the initiation of antibiotic therapy. However, this is not always the case especially in critically ill patients due to high rate of mortality associated with delayed treatment. In such patients, blood can be drawn prior to or after antibiotic therapy in order to avoid any fatality. Nevertheless, most blood culture media contain substances that help to inhibit the onslaught of antibiotics present in the collected blood for blood culture analysis. Different blood culture medium exist, and they each support anaerobic bacteria, facultative or aerobic bacteria.

AIM: To isolate and identify the presence of pathogenic bacteria in blood specimen as an aid in the diagnosis of bacteraemia and septicaemia.

MATERIAL/APPARATUS:  Blood specimen (about 10 ml), incubator, blood culture medium (e.g. Oxoid signal  blood culture system & medium), blood agar (BA), chocolate agar (CA), cystein lactose – electrolyte deficient (CLED) agar, inoculating loop, Bunsen burner, grease pencil, 70% ethanol, cotton wool. It should be noted that there are several blood culture medium available for blood culture. The type of blood culture medium used is left to the discretion of the researcher and the type of bacteria being isolated (anaerobic, aerobic or facultative organisms).

METHOD/PROCEDURE:

  1. Remove the protective cover from the top of the blood culture bottle.
  2. Clean/wipe the top of the blood culture bottle with a cotton swab of ethanol.
  3. Aseptically dispense the 10 ml of blood into the blood culture medium blood.
  4. Clean the top of the blood culture bottle medium with cotton swab of ethanol after dispensing or inoculating the blood specimen.
  5. Shake the blood culture bottle to mix the blood with the broth.
  6. Replace the culture signal device above the top of the blood culture bottle.
  7. Label the blood culture bottle with the patient’s name and number using a grease pencil.
  8. Incubate the inoculated blood culture bottle in the incubator at 37oC for 7 days.
  9. Examine the incubated blood culture bottle at intervals. Terminal subcultures can also be done before the 7 days elapses using CA, CLED, and BA agar plates.
  10. Perform a Gram stain to determine if the bacteria are either Gram positive or Gram negative.
  11. Then aseptically subculture an aliquot (the contents of the growth indicator device of the blood culture bottle) on relevant agar plates (e.g. CLED, BA, and CA) to look out for bacterial growth.
  12. Incubate the CLED & BA plates in the incubator at 37oC overnight.
  13. Incubate the CA plate in a carbon dioxide enriched atmosphere (e.g. anaerobic jar). The reason for this is to provide about 5 – 10% CO2 for the luxuriant growth of anaerobes present in the specimen.
  14. Examine plates for bacterial growth.
  15. Conduct biochemical tests and gram staining, and Perform antimicrobial susceptibility test only when a pathogen is successfully isolated and identified.

REPORTING OF THE RESULT:

Always report immediately a positive blood culture, and send a preliminary report of the stained smear and other useful test results to the physician in charge of the patient. This is very important as it is known that our blood does not have even a single normal microbial flora unlike other parts of the human body like the skin that are flooded with a wide variety of normal microbial flora. Presence of bacteria in the blood is indicative of a pathological/disease state. Report any pathogen that is isolated.

Bacteraemia is defined as the presence of bacteria in the blood. It is usually pathological (i.e. indicative of a serious disease) although transitory asymptomatic bacteraemia can occur during the course of many infections and after a surgical operation have been undertaken. Bacteraemia usually occurs in such diseases as: typhoid fever, endocarditis, and brucellosis.

Septicaemia is defined as a severe life – threatening bacteraemia. In septicaemic conditions, there is a large amount of bacteria in the blood, and these bacteria release toxins into the blood stream. These toxins trigger the production of cytokines, causing fever, low blood pressure, toxicity, and even collapse. A complication of septicemia is septic shock. Prompt treatment is essential. 

URINE CULTURE

Urine culture is performed in order to specifically identify organisms that may be causing a urinary tract infection (UTI). Urine in the bladder is often sterile as it is free from microorganisms. It becomes inundated with microorganisms (especially those of the micro-flora) when it leaves the bladder. UTI is very common in patients that are under catheterization and those having a poor personal hygiene. The source of urine sample for urine culture analysis can be mid-stream urine (MSU) or clean catch urine from a patient using a catheter (i.e. in critically ill patients who cannot naturally pass out urine).

AIM: To isolate and identify pathogenic bacteria from urine specimens of patient’s as an aid in the diagnosis of urinary tract infections.

MATERIAL/APPARATUS: Mid – stream urine (MSU) specimen, inoculating loop, Bunsen burner, incubator, cystein lactose – electrolyte deficient (CLED) agar, blood agar (BA), grease pencil.

METHOD/APPARATUS:

  1. Label the relevant agar plate (e.g. CLED and BA) with the patient’s name and laboratory number using a grease pencil.
  2. Mix the urine specimen by rotating the container.
  3. Using a sterilized inoculating loop, collect a loopful of the specimen and inoculate it on the culture media plates (CLED & BA).
  4. Incubate both plates in the incubator at 35oC overnight.
  5. Examine both plates for significant bacterial colonies.
  6. Perform biochemical testing (Citrate test and Indole test) and Gram staining.
  7. Perform antimicrobial susceptibility testing only when a pathogen has been isolated.

NOTE: It is not necessary to culture urine specimens that are microscopically and biochemically normal. Culture is only required when the urine specimen contains bacteria (as indicated by Gram smear), cells, casts, nitrite, or protein.

REPORTING OF THE RESULT:

Report bacterial numbers by counting the approximate number of colonies, and estimating the number of bacteria by colony forming unit per ml (CFU/ml). For bacterial counts, the following applies:

  • Bacterial counts less than 104/ml = Not significant.
  • Bacterial counts between 104 – 105/ml = Doubtful significance.
  • Bacterial numbers more than 105/ml = Significant bacteriuria.

A detection threshold of 105 CFU/ml of bacterial colonies resulting from a urine culture plate is significant of a bacteriuria.

Look particularly for E. coli, Klebsiella, Pseudomonas aeruginosa and Staphylococcus aureus e.t.c on the CLED plate. S. aureus cause haemolysis on BA, and this feature can be used to distinguish it from other bacteria. Other pathogenic microorganisms found in urine include Candida species, Streptococcus pneumoniae, Proteus mirabilis, Enterococcus species and Listeria monocytogenes.  

BA is used to isolate fastidious organisms e.g. Staphylococcus and Streptococcus that require additional nutrient like blood to grow.

CLED is a non – inhibitory differential medium which is specially used to isolate urinary pathogens from urine specimens. It allows the growth of both Gram positive and Gram negative pathogens. Lactose fermenting organisms appear yellow on CLED because the agar is bromothymol blue. CLED is electrolyte – deficient, and this prevents the growth of Swarming organisms like Proteus species.

SPUTUM CULTURE

Sputum culture is often recommended in the diagnoses of lower respiratory tract infection (e.g. pneumonia and pulmonary tuberculosis). Lower respiratory tract infections (LRTIs) are infections/diseases that occur below the voice box (larynx) i.e. in the bronchi and trachea. In this case, an induced or expectorated sputum specimen and not saliva is requested and obtained from the patient. The patient is asked to take a deep breath and then cough deeply to discharge what he or she coughed up into a sputum specimen collection container.

AIM: To isolate pathogenic bacteria from sputum specimens as an aid in the diagnosis of lower respiratory tract infections (e.g. pneumonia).

MATERIAL/APPARATUS: Sputum specimen, incubator, anaerobic jar, blood agar (BA), chocolate agar (CA), inoculating loop, grease pencil, face mask.

METHOD/PROCEDURE:

  1. Cover face with face mask to avoid splashing of aerosols that might emanate while working on the specimen.
  2. Label the BA and CA plates with the patients name and laboratory number using the grease pencil.
  3. Using a sterilized inoculating loop, collect a loopful of the sputum specimen.
  4. Inoculate it on both the CA and BA plates.
  5. Incubate the BA plate aerobically in the incubator at 37oC overnight.
  6. Incubate the CA plate anaerobically in an anaerobic jar at 37oC overnight.
  7. After incubation, examine both plates and look out for a significant growth of bacteria.
  8. Perform biochemical tests (oxidase test, catalase test, coagulase test) to identify the isolated bacteria.
  9. Perform antimicrobial susceptibility test only when a significant pathogen have been isolated

REPORTING OF THE RESULT:

Look especially for a significant growth of Streptococcus pneumonia, Haemophilus influenza, and Staphylococcus aureus and report.

NOTE: Sputum specimens should always be collected in leak-proof specimen containers and, such specimens should be treated with caution in order to avoid cross-contamination due to infected aerosol production which may occur during handling and processing of the specimen.

The culture and antimicrobial sensitivity testing of sputum specimens for Mycobacterium tuberculosis (the causative agent of tuberculosis – TB) are usually undertaken in a Tuberculosis reference center/laboratory that is normally outside the hospital environment. This is because aerosols from sputum specimens carrying M. tuberculosis can easily become airborne and cause infection. Though some hospitals have facilities for carrying out the microscopy, culture, and sensitivity of sputum specimens suspected to contain M. tuberculosis. It is also advisable to work under a laminar flow biological safety cabinet when handling and processing sputum specimens suspected to contain M. tuberculosis in order to prevent aerosols from the specimen from coming into contact with the microbiologist.

Anaerobic jar is used for the incubation of cultures in chocolate agar (CA) plates because the organisms for which this media (CA) is meant to isolate anaerobes/obligate aerobes and thus require a CO2 atmosphere for growth. The anaerobic jar increased CO2 tension which enhances the growth of the bacteria.

Illustration of an Anaerobic jar

MICROSCOPY OF SPUTUM SPECIMEN

AIM: To detect the presence of pus cells and predominant bacteria in sputum specimens as an aid in the diagnosis of lower respiratory tract infections like pneumonia or bronchopneumonia.

MATERIAL/APPARATUS: Sputum specimen, glass slide, microscope, immersion oil, inoculating loop, Bunsen burner, piece of stick.

METHOD/PROCEDURE:

  1. Transfer a purulent part of the sputum specimen to a clean glass slide using a sterilized inoculating loop or a piece of clean stick.
  2. Make a thin smear of the purulent sputum on the glass slide.
  3. Place the slide in a safe place away from dust for it to dry.
  4. Heat fix the smear on the slide by passing it thrice over a Bunsen burner flame.
  5. Perform the Gram staining technique.
  6. Allow the Gram stained slide to dry.
  7. Add a drop of immersion oil on the slide.
  8. Examine/Observe the stained slide under the microscope using the ×100/oil immersion objective lens.
  9. Look out for pus cells and predominant bacteria in the smear.

REPORTING OF THE RESULT:

Describe and report the appearance of the sputum specimen. The appearance of a sputum specimen can be described with any of the following parameters:

  • Purulent: Green – looking, mostly pus.
  • Mucopurulent: Green – looking with pus and mucus.
  • Mucoid: Mostly mucous.
  • Mucosalivary: mucous with a small amount of saliva.
  • Bloody: Sputum containing some amount of blood.

NOTE: The presence of blood in a sputum specimen can be due to Paragonimiasis caused by the parasite, Paragonimus westermani. P. westermani is a human lung fluke which affects the lungs.

Examine the slide carefully under the microscope and look especially for:

  • Capsulated Gram positive diplococcic (e.g. Streptococcus pneumoniae).
  • Gram positive cocci in groups (e.g. Staphylococcus aureus).
  • Capsulated Gram negative rods (e.g. Klebsiella pneumoniae).
  • Gram negative rods and cocco – bacilli (e.g. Haemophilus influenzae).
  • Gram negative diplococcic that are usually found in and between pus cells (e.g. Moraxella catarrhalis).

The result of a Gram stained smear of sputum specimen should be reported with utmost care and caution, as cocci, diplococcic, rods, and streptococci can also be seen in a normal sputum sample. This is because these organisms form part of the normal microbial flora of the human upper respiratory tract. The clinical presentation or diagnosis of the patient as given by the physician can also help in taking care of this.

CEREBROSPINAL FLUID (CSF) CULTURE

AIM: To isolate bacteria from cerebrospinal fluid (CSF) specimen as an aid in the diagnosis of cerebrospinal meningitis.

MATERIAL/APPARATUS: Cerebrospinal fluid (CSF) specimen, blood agar (BA), chocolate agar (CA), Bunsen burner, inoculating loop, incubator, anaerobic jar, MacConkey agar (MCA).

NOTE: The CSF specimen should be collected only by an experienced medical officer or health worker.  A lumber puncture is required to obtain the CSF specimen and should be done aseptically to avoid contamination. This is very important so as not to avoid introducing any contaminant into the CSF – which is a sterile fluid of the body. CSF is free from normal microbial flora and thus its collection and handling in the laboratory should be done aseptically. The examination/culture of CSF specimen should not be delayed like other specimens, and the results of tests should be immediately reported to the physician as soon as they become available. If the CSF specimen is from a newborn patient, MacConkey agar (MAC) should be used in addition with the blood agar (BA) and chocolate agar (CA). MAC and BA plates should be incubated aerobically while the CA plate should be incubated anaerobically. The CSF specimen comes to the microbiology laboratory in the string used for its collection, thus primary inoculants required for inoculation/culture should be obtained by releasing a drop on each of the culture media plates.

METHOD/PROCEDURE:

  1. Label the CA and BA plates with the patient’s name and laboratory number.
  2. Inoculate the CSF specimen on each of the CA and BA plates.
  3. Incubate the BA plate in the incubator at 37oC overnight.
  4. Incubate the CA plate in the anaerobic jar at 37oC overnight.
  5. After incubation, examine both plates for significant bacteria growth.
  6. Perform biochemical testing on significant bacterial isolates.
  7. Perform antimicrobial susceptibility testing only when a significant bacterium is isolated.

REPORTING OF THE RESULT:

Look especially for colonies that could be Escherichia coli or other coliform in the MAC plate and report. Look particularly for Neisseria meningitides, Streptococcus pneumonia, and Haemophilus influenzae on both the CA and BA plates and report appropriately.

MICROSCOPY OF CEREBROSPINAL FLUID (CSF)

The microscopical examination of CSF specimen has 2 basic aspects to it, viz:

  • Gram smear.
  • Cell count.
  • Differential count.

They are very important in the analysis of CSF specimen and each has its own role/significance in deciphering the presence of bacteria in a CSF. Their methods are expanded below. Before embarking on any of the above methods by which a CSF specimen can be analyzed microscopically, it is very important to also observe and report the appearance of CSF specimens first. The appearance of a CSF specimen can be reported based on whether the CSF specimen is:

  • Clear – normal CSF appears clear, bright, and colourless.
  • Cloudy or purulent – indicates the presence of pus cells that is suggestive of pyogenic bacterial meningitis.
  • Bloody – blood in CSF specimen may be due to a bloody/traumatic lumbar puncture.
  • Contains clots – this indicates a high protein concentration with increased fibrinogen.
  1. GRAM SMEAR OF CSF SAMPLE

AIM: To detect the presence of pus cells and bacteria in cerebrospinal fluid (CSF) specimen.

MATERIAL/APPARATUS: CSF specimen, Gram staining reagents, microscope, glass slide, immersion oil, Bunsen burner.

METHOD/PROCEDURE:

  1. Place a drop of the CSF specimen on a clean glass slide.
  2. Make a thin smear of the CSF specimen.
  3. Allow to dry in a secure place to prevent it from dust.
  4. Heat fix the smear by passing it thrice over the blue flame of a Bunsen burner.
  5. Perform the Gram staining technique.
  6. Allow the Gram stained slide to dry.
  7. Place a drop of immersion oil on the slide.
  8. View the slide under the microscope using the ×100/oil immersion objective lens

REPORTING OF THE RESULT:

Look for Gram negative intracellular diplococcic, Gram negative rods, Gram positive diplococcic, and pus cells and report. If the Gram smear contains bacteria and pus cells, inform the physician about it immediately. Culture the CSF specimen once the Gram smear proves positive for bacteria and pus cells. In cases of an emergency treatment when the patient is given antibiotic treatment before the CSF specimen is collected, it is possible and more difficult to detect bacteria in a Gram stained smear and to isolate bacteria from culture.

  1. CELL COUNT OF CSF SAMPLE

AIM: To determine whether the cells found in a CSF specimen are pus cells or lymphocytes. And to count the white blood cells (WBC’s) present in the CSF.

MATERIAL/APPARATUS: CSF specimen, microscope, cover slip, improved neubauer chamber, Pasteur pipette or bulb pipette, Turk’s solution, test tube test tube rack.

METHOD/PROCEDURE:

  1. Assemble the improved neubauer chamber, making sure that both the chamber and cover slip are completely clean.
  2. Mix the CSF specimen. This is done by rotating the string used in obtaining it.
  3. Carefully fill the improved neubauer counting chamber with the CSF specimen, making sure not to allow the fluid to flow into the channels on each side of the chamber.
  4. Wait for about 2 minutes for the cells to settle.
  5. Focus the cells on the chamber under the microscope using the ×10 and ×40 objective lens. Make sure that the condenser iris of the microscope is sufficiently closed in order to give a good contrast.
  6. Count the cells in 4 of the large squares indicated with the letter ‘W as shown in the figure below.

NOTE: Use Turk’s solution to dilute bloody CSF specimen before filling the improved neubauer chamber. They are 2 ways to doing this dilution depending on the amount of the blood in the CSF.

  1. One in five dilutions (1:5): This dilution is made if the CSF specimen contains much blood. It is done by placing 4 drops of Turk’s solution plus 1 drop of the CSF specimen in a clean test tube.
  2. One in two dilutions (1:2): This dilution is made if the CSF specimen does not contain much blood. It is done by placing 1 drop of Turk’s solution plus 1 drop of the CSF specimen in a clean test tube.

NOTE: The essence of diluting a bloody CSF specimen before performing any analysis on it is to ‘lyse/breakdown the red blood cells (RBC’s) in the specimen ’. The RBC’s makes it unsuitable or difficult to count the white blood cells (WBC’s).

Improved Neubauer countingchamber

REPORTING OF THE RESULT:

Report the appearance of the CSF specimen based on whether it is cloudy, clear, purulent or bloody. Count the white blood cells in 5 of the large squares of the improved neubauer chamber as shown in the diagram above, and report the number of cells by multiplying the number of WBC’s counted with the number of dilution made.  An example on how to do this is as follows:

  • For 1 in 2 CSF dilutions: If for example you counted 200 WBC’s, it will be 200 × 2 = 400.

Report the cell count as: 400 × 106 cell/mm3

  • For 1 in 4 CSF dilutions: If for example you counted 80 WBC’s, it will be 80 × 4 = 320.

Report the cell count as: 320 × 106 cell/mm3/

  1. DIFFERENTIAL COUNT OF CSF SAMPLE

AIM: To provide information on the different white blood cells (neutrophils, basophils, eosinophils, lymphocytes, monocytes) present in the CSF specimen.

MATERIAL/APPARATUS: CSF specimen, methylene blue stain, microscope, glass slide, water, Bunsen burner, inoculating loop, immersion oil.

METHOD/PROCEDURE:

  1. Place a drop of the CSF specimen on a clean glass slide.
  2. Spread using a sterilized inoculating loop and allow to dry.
  3. Heat fix the smear on the glass slide.
  4. Flood the slide with methylene blue stain.
  5. Allow for 5 minutes. Then wash off the methylene blue stain with water.
  6. Allow the slide to dry completely.
  7. Add a drop of immersion oil on the slide.
  8. View the slide under the microscope using the ×100/oil immersion objective lens.
  9. Systematically examine the slide and count the different white cells seen in each field.

REPORTING OF THE RESULT:

Report the number of the different white cells seen.

NOTE: Differential white cell count can be performed using 3 types of stains:

  • Methylene blue stain.
  • Giemsa stain.
  • Leishma stain

SEMINAL FLUID (SEMEN) CULTURE

AIM: To isolate pathogenic bacteria from semen specimens as an aid in the diagnosis of infertility in male.

MATERIAL/APPARATUS: Semen specimen, grease pencil, incubator, anaerobic jar, Bunsen burner, inoculating loop, blood agar (BA), chocolate agar (CA).

METHOD/PROCEDURE:

  1. Label the CA and BA plates with the patient’s name and laboratory number using the grease pencil.
  2. Aseptically culture the semen specimen on both the CA and BA plates.
  3. Incubate the BA plate in the incubator at 37oC overnight.
  4. Incubate the CA plate in the anaerobic jar at 37oC overnight.
  5. Perform biochemical test to identify the isolated bacteria.
  6. Perform antimicrobial sensitivity test only when a significant bacteria is isolated.

REPORTING OF THE RESULT:

Significant bacteria growth should be reported. The culture of semen specimen is carried out first before any microscopical analysis in order to avoid contaminating the specimen.

SEMEN MICROSCOPY

AIM: To determine any abnormality in a seminal fluid (semen) as an aid in the diagnosis of male infertility.

MATERIAL/APPARATUS: Semen specimen, microscope, test tube, test tube rack, glass slide, cover slip, semen diluting fluid (sodium bicarbonate formalin), improved neubauer counting chamber, bulb pipette or Pasteur pipette, calibrated cylinder (5 ml).

The microscopy of a semen specimen involves the following procedures:

  • Determination of the semen volume.
  • Determination of the semen viscosity.
  • Determination of the motility of the sperm cells in the semen specimen.
  • Determination of the morphology of the sperm cells in the semen specimen.
  • Sperm cell count.

In addition to the above parameters, the time of production, time of collection, time of examination, and the appearance of the semen specimen is taken into cognizance as well. The appearance of a semen specimen can be grayish – white e.t.c. These parameters are also reported together with other analysis carried out.

SEMEN VOLUME

The volume of a semen specimen can be determined by emptying or pouring the semen specimen into a calibrated cylinder that is about 5 ml. The volume of normal semen is between 2 – 5 ml and above.

SEMEN VISCOSITY

A semen specimen can be viscous, non – viscous, slightly viscous or highly viscous. Due to the presence of fibrinolysin in the semen, it usually becomes liquefied within 1 hour. You can determine how viscous a semen specimen is by touching it with a sterile inoculating loop or stick and raising it up. Normal semen is thick and viscous when ejaculated or produced.

SPERM CELL MOTILITY

The procedure for doing this is as follows:

  • Place one drop of well mixed semen on a clean glass slide.
  • Cover with a cover slip.
  • Focus the slide using ×10 and ×40 objective lens of a microscope. Ensure that the condenser iris of the microscope is sufficiently closed in order to give a good contrast while viewing.
  • Examine several fields of the slide and report your result.

REPORTING THE RESULT:

A sperm cell can be actively motile (rapid and progressive) or weakly motile (slow and non – progressive). Count a total of 100 spermatozoa/sperm cell, and note out of the 100 how many spermatozoa are motile. Record the percentage that is motile and non – motile. Normal semen should have more than 50% motile spermatozoa within 60 minutes of ejaculation.

SPERM CELL MORPHOLOGY

The procedure for doing this is as follows:

  • Place a drop of well mixed semen on a clean glass slide.
  • Make a thin smear of the semen on the slide.
  • Heat fix the smear.
  • Stain the smear with either Giemsa stain or methylene blue stain.
  • Allow for 5 minutes.
  • Wash off with water after 5 minutes and allow to dry.
  • Add a drop of immersion oil on the slide.
  • Focus with ×100 objective lens.
  • Examine the different fields for any abnormality in the morphology of the spermatozoa.

REPORTING OF THE RESULT:

Estimate the number of spermatozoa showing normal morphology and abnormal morphology.

Normal spermatozoa measures about 50 – 70 µm in length, and each consists of an oval – shaped head (with acrosomal cap), a short middle piece, and a long tail. In normal semen, at least 50% of spermatozoa should show normal morphology.

Schematic illustration of a sperm cell

SPERM CELL COUNT

The procedure for doing this is as follows:

  • Make a 1 in 10 dilution of the semen specimen using a calibrated test tube and semen diluting fluid as follows:

Place 9 drops of the semen diluting fluid (sodium bicarbonate formalin) in a clean test tube standing on a test tube rack. Add a drop of well mixed semen specimen, and mix well. Avoid bubble production when mixing the semen and the diluent. In this way, a 1 in 10 dilution of a semen specimen is made. I in 10 dilutions is made for small semen specimens. For large semen specimens, 1 in 100 dilutions is made. Counting of spermatozoa in a semen specimen without dilution will be very difficult because the sperm cells are still alive and moving. This is why the semen diluting fluid is used so that they can inactivate or kill the spermatozoa, making the count easy.

  • Using a Pasteur’s pipette, fill an improved neubauer counting chamber with the well mixed diluted semen specimen.
  • Wait for about 4 – 5 minutes for the sperm cells to settle, before viewing.
  • View the counting chamber with ×10 objective lens of a microscope. Make sure the condenser iris of the microscope is sufficiently reduced in or order to give a good contrast.
  • Count the sperm cells in each of the 4 fields of the counting chamber.

REPORTING THE RESULT:

The formula for counting and reporting a sperm cell count is thus:

Number of sperm cells counted × 104 × diluting factor. The unit is: Sperm cells/ml.

Normal sperm cell count is: above 20 × 106 sperm cells/ml.

Abnormal sperm cell count is: below 20 × 106 sperm cells/ml.

VAGINAL SWAB CULTURE

AIM: To isolate pathogenic bacteria from urogenital specimens (e.g. vaginal swab, urethral swab) as an aid in the diagnosis of Candidiasis and bacterial vaginosis or bacterial infections of the ureter, vagina, and cervix.

MATERIAL/APPARATUS: Urogenital specimen, blood agar (BA), cystein lactose – electrolyte deficient (CLED) agar, incubator, chocolate agar, grease pencil, Bunsen burner, inoculating loop.

METHOD/PROCEDURE:

  1. Label the culture plates (CLED, BA, and CA) with the patient’s name and hospital number using the grease pencil.
  2. Aseptically inoculate the urogenital specimen on the CLED, BA, and CA plates.
  3. Incubate the CLED and BA plates in the incubator at 37oC overnight.
  4. Incubate the CA plate in an anaerobic jar at 37oC overnight.
  5. Perform biochemical testing on significant bacterial growth.
  6. Perform antimicrobial susceptibility testing on significant bacterial isolates.

REPORTING OF THE RESULT:

Report only significant bacterial growth after performing biochemical tests to identify the isolated organisms. Bacteria likely to be isolated from urogenital specimens include: Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli,, Proteus, Enterococcus, Neisseria gonorrhoeae. N. gonorrhoeae is isolated from urogenital specimens using a selective enriched culture medium media called Thayer Martin medium.

MICROSCOPY OF UROGENITAL SPECIMENS

The microscopical examination of urogenital specimens may involve one of the following procedures:

  • Gram smear.
  • Wet preparation.

NOTE:  Most urogenital specimens come in swab stick form. A sterile non – toxic cotton swab stick is used to obtain or collect urogenital specimens from patients.

  1. GRAM SMEAR OF UROGENITAL SPECIMENS

AIM:  To detect the presence of pus cells, bacteria, and yeast cells from urogenital specimens.

MATERIAL/APPARATUS: urogenital specimen, glass slide, immersion oil, microscope, Gram staining reagents.

 METHOD/PROCEDURE:

  1. Gently roll the swab stick containing the urogenital specimen on a clean glass slide. In this way, a smear of the urogenital specimen is made, and it will also help to avoid any damage that will be done to the pus cells containing the bacteria.
  2. Heat fix the smear on the slide.
  3. Perform Gram staining technique.
  4. Allow the Gram stained slide to dry.
  5. Add a drop of immersion oil on the slide.
  6. Examine the slide with the ×10/oil immersion objective of a microscope.
  7. Look out for pus cells and bacteria as you view the slide.

REPORTING OF THE RESULT:

  • For Gram smear from suspected gonorrhea: Look for pus cells containing Gram negative diplococcic or intracellular Gram negative diplococci. If the pus cells are damaged, the Gram negative diplococci will be extracellular i.e. placed outside the pus cells. This is presumptive diagnosis for gonorrhea especially in males that are symptomatic to the infection. Gram stained smear does not always indicate the presence of gonorrhea in women because women are asymptomatic to the infection. Thus culture is required to diagnose gonorrhea in women.
  • For vaginal smear from candidiasis or bacterial vaginosis patients: Look for large Gram positive yeast cells that could be Candida albicans or other species of Candida. This is indicative of the presence of candidiasis infection. Also look out for Clue cells (epithelial cells with adhering Gram negative short bacilli and Gram variable coccobacilli) that could be Gardnerella vaginalis. This is indicative of bacterial vaginosis.
  1. WET PREPARATION OF UROGENITAL SPECIMENS

AIM: To detect the presence of yeast cells, Trichomonas vaginalis, pus cells, red blood cells (RBC’s) from urogenital specimens as an aid in the diagnosis of Trichomoniasis and Vaginal candidiasis.

MATERIAL/APPARATUS: Urogenital specimen (e.g. vaginal swab), test tubes, test tube rack, physiological (normal) saline, glass slide, cover slip, microscope, bulb pipette.

METHOD/PROCEDURE:

  1. With the bulb pipette, obtain about 1 – 2 ml of normal saline.
  2. Dispense the normal saline into a clean test tube standing on a test tube rack.
  3. Immerse the cotton swab stick containing the urogenital swab into the test tube.
  4. Allow to stand for about 60 seconds.
  5. Bring out the swab stick and make a wet preparation on a clean glass slide. Ensure that only a thin preparation is made.
  6. Cover the preparation with a cover slip.
  7. Examine slide with ×40 objective lens of a microscope. Ensure that the condenser iris of the microscope is sufficiently closed in order to give a good contrast.

NOTE: Wet preparation of urogenital specimens are usually done or carried out after Gram smear and culture must have been undertaken. This is to ensure that the specimen is not contaminated before culturing and Gram smearing.

REPORTING OF THE RESULT:

Trichomonas vaginalis is motile. Look out for motile T. vaginalis and report. Look out for yeast cells, RBC’s, and pus cells.

THROAT SWAB CULTURE

Throat swab culture is usually in the diagnosis of upper respiratory tract infections (URTIs) e.g. pharyngitis, Otitis media and epiglottitis. URTIs are infections of that begins from the larynx to the nostrils and, it also involves other communicating cavities that are connected to the respiratory tract and nostrils e.g. the middle ear and its sinuses.     

AIM: To isolate pathogenic bacteria from throat swab specimen as an aid in the diagnosis of upper respiratory tract infections.

MATERIAL/APPARATUS: Throat swab, blood agar (BA), chocolate agar (CA), cysteine lactose – electrolyte deficient (CLED) agar, inoculating loop, anaerobic jar, incubator, grease pencil, Bunsen burner.

METHOD/PROCEDURE:

  1. Label the CA, BA, and CLED plates with patient’s name and laboratory number using the grease pencil.
  2. Inoculate the throat swab specimen on the CLED, BA, and CA plates.
  3. Incubate the CLED and the BA plates in the incubator at 37oC overnight.
  4. Incubate the CA plate in the anaerobic jar at 37oC overnight.
  5. Examine the culture plates for significant bacterial growth after incubation.
  6. Perform biochemical tests on significant bacteria pathogens isolated.
  7. Perform antimicrobial susceptibility testing only on significant bacteria isolates.

REPORTING OF THE RESULT:

Cultures of throat swabs are most reliable if inoculated promptly after collection. The normal throat flora includes an abundance of viridians Streptococci, Neisseria, diphtheroids, Staphylococci, small Gram negative rods, and other organisms.

The reporting of a throat swab culture should be done with utmost care so as not to report a normal bacterial flora found in the throat, giving that the oral cavity is flooded with a wide variety of bacteria that are resident in the mouth.

Microscopical analysis though of little significance can also be used in investigating an upper respiratory tract infection. It is not often used because of the predominance of Streptococci in all throats

SATELLITISM TEST

Satellitism test is a culture-based test which is used to identify Haemophilus influenzae in the laboratory. Haemophilus influenzae is a fastidious bacterium that requires both X factor (haemin) and V factor (NAD or NADP) for growth. Blood agar lacking any of these growth factors will not support most strains of H. influenzae. When grown in blood culture media, S. aureus produce NAD as a metabolic by product. For that reason, species of Haemophilus may grow very closely to the colonies of S. aureus when streaked on sheep blood agar.  S. aureus produces NAD (V factor), a key requirement for the growth of H. influenzae. This phenomenon is known as satelliting, and the test is called satellitism.  

AIM: The aim is to confirm and identify H. influenzae strain.  

MATERIALS/APPARATUS: Suspect colonies of H. influenzae, sheep blood agar plates, incubator, S. aureus culture, anaerobic jar (for provision of mild CO2 tension), peptone water, tryptic soya agar, swab sticks, inoculating loop.

Illustration of satellitism test

METHOD/PROCEDURE:

  1. Mix a loopful of suspected Haemophilus colonies in about 2 ml of sterile peptone water. Ensure that none of the Chocolate agar medium (from which the Haemophilus was obtained from) is transferred into the sterile peptone water tube.
  2. Aseptically inoculate the suspect Haemophilus colonies on a plate of sheep blood agar and tryptic soya agar.
  3. Using a sterile loop, streak a pure culture of aureus across each of the inoculated sheep blood agar and tryptic soya agar plates.
  4. Incubate both plates in the incubator at 35-37oC overnight (in the presence of mild CO2 tension).
  5. Examine the culture plates for growth and satellite colonies of influenzae.

REPORTING OF THE RESULTS:

The suspect colonies are confirmed as H. influenzae if growth is seen in the sheep blood agar but not in the tryptic soya agar, and if the colonies of H. influenzae near the column of S. aureus growth are larger than those farthest from it. Colonies growing farther from the S. aureus column are referred to as “satellite colonies”, which depicts the name of the test satellitism. Colonies of streptococcus, Neisseria, and Diphtheroids also exhibit the satellitism phenomenon.

Note: If satellite colonies are present on both the sheep blood agar and tryptic soya agar plates, then the organism is a Haemophilus species that requires only the V factor (NAD or NADP) for growt

PARASITOLOGY

Parasitology is simply defined as the study of parasites. But for the purpose of this work, only medically important parasites (i.e. parasites that cause disease in humans) shall be considered here. Parasites are organisms that depend entirely on another organism known as the host, for all or part of its life cycle and metabolic requirements. Specimens encountered in the clinical Parasitology laboratory includes: feaces, urine, liver aspirates, blood, bile, sputum, corneal scrapings, duodenal aspirates and tissues. Though majority of the analysis or investigations carried out with these specimens are normally undertaken microscopically, specialist Parasitology laboratories (and reference laboratories) also incorporate serology (e.g. ELISA) and polymerase chain reaction (PCR) techniques in their parasitological investigations.

A parasite is an organism that is able to live in or on the body of another organism called a host and cause disease. The relationship that exists between a host and a parasite (in which only the parasite benefit) is called parasitism. There are two types of parasite: ectoparasites and endoparasites. Ectoparasites are parasites that live on the body of their host. Example of ectoparasites include: lice and tick. Endoparasites are parasites that live inside the body of their host. Example of endoparasites include: worms, Plasmodium and Trypanosome. Parasites (endoparasites and ectoparasites) are implicated in a variety of tropical diseases including malaria and diarrhea and, they present a major public health issue in most parts of the world especially in the developing countries where sanitation, water supply and environmental sustainability is still being improved upon.

The parasitological unit of the microbiology laboratory is mainly responsible for the macroscopic and microscopic investigation of parasites, their cysts, and ova’s in the specimen of patient’s. The specimens encountered in the parasitological unit of the microbiology laboratory are: urine, stool (feaces), blood, and skin snip. An essential procedure in the diagnosis of all parasitic infections is the microscopic recognition or identification of ova or larvae of the infecting parasites in feaces, blood, urine, or tissues of patients as they report to the hospital with one helminthic infection or the other. 

PARASITES OF MEDICAL IMPORTANCE ENCOUNTERED IN THE PARASITOLOGY LABORATORY

PARASITE COMMON NAMES DISEASE CAUSED/ORGAN ATTACKED 
Wuchereria bancrofti Microfilaria Bancroftian filariasis
Ancylostoma duodenale Hookworm Parasitosis
Fasciola hepatica Liver fluke Liver
Loa loa Microfilaria Loiasis
Ascaris lumbricoides Roundworm Parasitosis
Onchocerca volvulus Microfilaria River blindness
Necator americanus Hookworm Parasitosis
Paragonimus westermani Lung fluke Paragonimiasis
Trypanosome cruzi Blood parasite Chagas disease
Giardia lamblia Intestinal protozoa Giardiasis
Balantidium coli Intestinal parasite Balantidial dysentery
Entamoeba histolytica Intestinal parasite Amoebic dysentery
Isospora belli Intestinal coccidia Diarrhea in AIDS patients
Cyclospora cayetanensis Intestinal coccidia Diarrhea in AIDS patients
Cryptosporidium parvum Intestinal coccidian Diarrhea in AIDS patients
Trichuris trichiura Whipworm Trichuriasis
Strongyloides stercoralis Dwarf threadworm Strongyloidiasis
Schistosoma species Intestinal parasites Schistosomiasis
Fasciolopsis buski Intestinal fluke Fasciolopsiasis
Taenia sanigata Beef tapeworm Taeniasis
Taenia solium Pork tapeworm Taeniasis
Diphyllobothrium latum Fish tapeworm Diphyllobothriasis
Trypanosoma brucei gambiense Blood parasite Sleeping sickness (West African trypanosomiasis)
Trypanosoma brucei rhodesiense Blood parasite Sleeping sickness (East African trypanosomiasis)
Hymenolepsis nana Dwarf tapeworm Diarrhea
Leishmania species Kala-azar Leishmaniasis
Dracunculus medinensis Guinea worm Dracunculiasis
Toxoplasma gondii Toxoplasma Toxoplasmosis
Plasmodium species Blood parasite Malaria

SOME OF THE MAJOR PROCEEDINGS UNDERTAKEN IN THE PARASITOLOGICAL UNIT OF THE MICROBIOLOGY LABORATORY INCLUDE:

  • Stool analysis
  • Urine analysis
  • Occult blood test (OBT)
  • Skin snip
  • Wet mounting
  • Concentration/floatation technique
  • Urine chemistry

CONCENTRATION METHODS IN THE PARASITOLOGY LABORATORY

Concentration techniques are used to concentrate the eggs/ova of parasites and the cysts/trophozoites of protozoa in feacal samples especially when they occur in small amounts in the sample being examined. This method does not detect the motile forms of parasites in feacal samples; thus the microscopy of the sample should first be undertaken before carrying out any concentration technique using the same sample. There are three concentration methods available in the Parasitology laboratory:

  1. Sedimentation method: This technique makes use of a filter paper strip to concentrate the larvae of parasites. The filter paper is spread with the feacal specimen and then inserted into a tube containing clean boiled water, sealed with cotton wool and allowed to stand for about 4-6 days. Any larvae present in the feacal specimen will travel against the water current and settle at the bottom of the test tube. The sedimentation concentration method is used to investigate the larvae of parasites such as Strongyloides stercoralis.
  2. Formaldehyde method: This technique is divided into two parts: the formaldehyde-ether sedimentation technique and the formaldehyde-detergent sedimentation technique. Both of these techniques make use of centrifugation and different concentrations of formalin to concentrate the eggs/ova of parasites but the formaldehyde-ether sedimentation technique also adds ether in its methodology. The presence of formaldehyde helps to preserve any parasite which may be present in the feacal specimen.
  3. Floatation method: The floatation concentration technique involves the mixing of the feacal specimen in a saturated solution of sodium chloride. The saturated sodium chloride solution allows the eggs/ova of the parasites in the feacal sample to float to the surface of the solution from where they can be conveniently harvested. Floatation method is appropriate for detection of the eggs of Ancylostoma duodenale, Ascaris lumbricoides, and Taenia

STOOL ANALYSIS

Stool analysis is the microscopic and macroscopic examination of a stool/feacal specimen to decipher the presence of parasites ova or larvae in them. The reasons for examining feacal specimens include:

  • To identify the parasitic causes of blood and mucus in feaces and differentiate amoebic dysentery from bacterial dysentery.
  • To detect occult/hidden blood in feaces as a way of providing evidence for peptic ulcer.
  • To identify intestinal parasitic infections that requires treatment.
  • To detect the presence of mucus flecks in the stool specimen in cases of cholera infections.
  • To detect serious hookworm infection in patients with anaemia

The macroscopic examination of stool specimens involves the following:

  • Appearance of the stool specimen. The appearance of the stool specimen under investigation is taken into cognizance with respect to its colour, as to whether it is pale, black or brown.
  • Consistency of the stool specimen. The consistency of the stool specimen refers to the nature of the feaces, whether it is formed, semi-formed, unformed, or watery.
  • Presence of worms in the stool specimen.
  • Presence of blood, mucus, or pus in the stool specimen. It is noteworthy that the presence of blood on only the surface of a stool specimen does not necessarily indicate the presence of infections, as this may be due to anal or rectal bleeding caused while passing out the feaces.

The microscopic examination of stool specimens in the parasitological unit involves:

  • Wet mounting/preparation of the stool specimen.
  • Concentration/floatation technique.

WET MOUNTING/PREPARATION

Wet mounting is a direct microscopic examination of feacal specimens.

AIM: To detect the presence of motile parasites, white blood cells (WBC), red blood cell (RBC), casts, cysts, and eggs of parasites, as an aid in the diagnosis of helminthic infections.

MATERIALS/APPARATUS: Stool specimen, binocular microscope, glass slides, cover slip, bulb pipette physiological/normal saline, applicator stick, cotton wool, discard jar.

METHOD/PROCEDURE:

  1. Place 2 drops of normal saline on a clean glass slide using a bulb pipette.
  2. Using the applicator stick, collect a small amount of the feaces and mix it with the normal saline on the glass slide.
  3. As you emulsify/mix the feacal specimen and the normal saline together, make sure that a thin smooth preparation is made. Otherwise, the preparation will be too thick, making it difficult to detect and identify parasites or any of its components present in the specimen.
  4. Cover the preparation/mixture on the glass slide with a cover slip.
  5. Examine the prepared slide under the microscope using ×10 objective lens to focus, and then ×40 objective lens to confirm.

Note:  The condenser iris of the microscope should be closed sufficiently before viewing in order to get a very good contrast. Also, always make sure that you examine several fields of the slide before making your final report, so as to clear all doubts.

REPORTING OF THE RESULT:

Report the appearance of the stool specimen first based on its colour and consistency. Report the number of larvae and egg/ova found as: scanty, few, moderate number, many, or very many. The presence of WBC, RBC, or cysts is also reported using + sign, and if not found in the specimen; they are reported as “nil”.

FEACAL CONCENTRATION TECHNIQUE (FLOATATION)

AIM: To concentrate parasites, their eggs, and cysts found in feacal specimens as an aid in the diagnosis of parasitic infections.

PRINCIPLE: The principle of floatation technique of feacal sample concentration is based on the use of saturated sodium chloride solution which has a specific gravity (relative density) that is higher than that of the parasites, their cysts, and eggs; thus allowing them to float to the surface of the brine solution after been emulsified with it and left to stand undisturbed for a given time period.

MATERIALS/APPARATUS: Stool specimen, saturated sodium chloride solution, glass slide, cover slip, test tube, applicator stick, binocular microscope, bulb pipette, test tube rack, timer.

METHOD/PROCEDURE:

  1. Fill a clean test tube standing on a test tube rack about half filled with the saturated sodium chloride solution.
  2. Collect a good portion of the stool specimen using the applicator stick and emulsify/mix it with the saturated sodium chloride solution in the half filled test tube.
  3. Fill the test tube to the brim with the saturated sodium chloride solution using the bulb pipette.
  4. Carefully place a clean cover slip on top of the test tube. Avoid trapping air bubbles as you do this.
  5. Allow the test tube to stand undisturbed on the test tube rack for about 15 minutes. NOTE: This time is to allow the parasites, their cysts, and eggs to float to the surface of the saturated sodium chloride solution so that they can be properly harvested.
  6. Carefully lift/pull the cover slip straight upwards.
  7. Place the cover slip downwards on a clean glass slide.
  8. Examine the glass slide using the ×10 objective lens and ×40 objective lens of the binocular microscope.

Note:  The condenser iris of the microscope should be closed sufficiently before viewing in order to get a very good contrast. Also, always make sure that you examine several fields of the slide before making your final report, so as to clear all doubts.

REPORTING OF THE RESULT:

Count the number of parasites found and report by their name. Also, the cysts, eggs, WBC, and RBC found are reported as well and if none of these is discovered during the examination, they are reported as “nil”.

It is noteworthy that floatation technique is one of the types of concentration technique which is used to concentrate feacal specimens in the clinical microbiology laboratory. The other concentration techniques are: sedimentation technique, formol ether concentration technique, and zinc sulphate concentration technique.

OCCULT BLOOD TEST (OBT)

AIM: To detect the presence of occult (hidden) blood in stool specimens as an aid in the diagnosis of infections that cause bleeding lesions of gastrointestinal tract (GIT), e.g. peptic ulcer, carcinoma, or diverticulosis.

MATERIAL/APPARATUS: Stool specimen, HEMA screen test paper, HEMA screen developing solution, timer. NOTE: Both HEMA screen test paper and HEMA screen developing solution are components of HEMA SCREEN test kit used for occult blood test. Other methods of detecting occult blood in stool specimen include: aminophenazone test, and the immunological test using ready – made reagents in kit tests.

PRINCIPLE: The principle behind the use of HEMA SCREEN test kit in the detection of occult blood in stool specimen is thus: the HEMA SCREEN test paper is made from a plant (tree) called Guaicidium offinacle, and this paper contains a component called guaiacum or guaiac – a chromogen. Haemoglobin (a component of blood) reacts in a similar way to peroxidase enzymes, i.e. they catalyze the transfer of an oxygen atom from a peroxidase such as hydrogen peroxide (H2O2) to a chromogen such as guaiacum, 2, 6 – dichlorophenolindophenol or aminophenazone. Oxidation of the chromogen is shown by the production of a blue – green, or pink colour.

METHOD/PROCEDURE:

  1. Collect a portion of the stool specimen using the applicator stick. Make sure that you collect from the middle of the specimen and not just from the surface only.
  2. Open the HEMA SCREEN test paper and spread the stool specimen collected on the test area provided. Ensure that only a thin smear is made on the test area.
  3. Add 2 drops of the HEMA SCREEN peroxidase developing solution onto the smeared stool specimen on the test area.
  4. Cover the test paper and press with your hand, and open the reverse side of the test paper after 30 seconds to read the result

REPORTING OF THE RESULT:

Presence of a blue – green colouration is a positive result, and it shows that the stool specimen contains occult blood. Absence of a blue – green colouration is a negative result, and it shows that the stool specimen does not contain occult blood.

NOTE: Occult blood test (OBT) is very important because it detects occult/hidden blood which occurs in the GIT when bleeding is chronic (slow), and blood is passed into the feaces in small amounts that cannot be detected. This blood is not recognized when looking at the feacal sample physically, and is thus called occult (hidden) blood. That is why OBT is needed to detect such blood and its breakdown products that pass into the feaces undetected.

It is also very important to tell the patient to deviate from eating vegetables and meat for at least two days prior to collecting the stool specimen. This is because the duo (meat and vegetables) contain peroxidase and thus will react with the test paper to give a false positive result.

SKIN SNIP TEST

AIM: To detect and identify the microfilariae of Onchocercia volvulus in skin snips as an aid in the diagnosis of Onchocerciasis.

Technique: There are two techniques to performing this test, viz: the slide technique and the test tube technique, but only the slide technique shall be expanded here as it is the most common and widely method of detecting microfilariae of Onchocercia volvulus.

MATERIAL/APPARATUS: Skin snip, microscope, glass slide, cover slip, normal saline, bulb pipette, scalpel blade, 70% ethanol, cotton wool.

METHOD/PROCEDURE:

  1. Clean the skin with 70% of ethanol using a cotton wool and allow to dry.
  2. Make a short quick stroke or cut at the skin where the specimen is to be collected using a scalpel blade.
  3. Cut of the piece of skin with a different scalpel blade, and place the skin snip obtained on a glass slide already containing 2 drops of normal saline.
  4. Cover the glass slide with a cover slip.
  5. Examine the slide under the microscope using ×10 and ×40 objective lens.

NOTE: A bloodless skin snip should be collected so as not to contaminate the specimen.

REPORTING OF THE RESULT:

The presence of any microfilariae is reported, and when not found it is reported as absent. A nodule present on the skin area where the skin snip is collected is also reported.

Onchocerciasis is a major health and socioeconomic problem, especially in endemic areas in Africa. It is caused by Onchocercia volvulus, and its clinical features are the formation of nodules, inflammatory dermatitis which is usually accompanied by intense irritation & raised papules on the skin, and inflammatory reactions in the eye leading to blindness. Onchocerciasis is also thought to be a risk factor for epilepsy.

Blindness is one of the most serious complications of Onchocerciasis, and it occurs when microfilariae of O. volvulus in the skin of the face migrate into the eye. There is redness and irritation of the eye. Progressive changes caused by inflammatory reactions around damaged and dead microfilariae can cause sclerosis keratitis which can lead to blindness. O. volvulus microfilariae can also be found in urine, blood, and in most body fluids. These specimens especially blood can also be obtained from affected patients for microbiological examination. Snip means a cut with scissors or blade using short quick strokes.

URINE MICROSCOPY

AIM: To detect the presence of RBC’s, WBC’s, casts, yeast cells, crystals, bacteria in urine deposits as an aid in the diagnosis of urinary tract infections (UTI’s).

TECHNIQUE: The technique used in urine microscopy is called wet mounting/preparation.

MATERIAL/APPARATUS: Urine specimen, microscope, glass slide, cover slip, test tube, test tube rack, discard jar, grease pencil, centrifuge.

METHOD/PROCEDURE:

  1. Describe the appearance of the urine specimen first by reporting its colour (e.g. pale yellow, yellow, and amber, bloody or smoky brown) and turbidity (i.e. whether it is clear or cloudy).
  2. Transfer about 10 ml of the urine specimen into clean test tube. In cases of many urine specimens, label the test tubes using grease pencil to avoid any chaos that might emanate in the course of the analysis.
  3. Place the test tube (s) containing the urine specimen into the centrifuge machine and centrifuge at 400 rpm for 5 minutes. This is done in order to sediment the cells and other materials/particles sought for in the urine specimen.

RPM means “Revolutions per Minute”.

  1. Pour the supernatant of the test tube content after centrifuging, into a discard jar. This is done by completely inverting/holding the test tube at an angle of 90o over the discard jar to get rid of the supernatant.
  2. Remix the sediment remaining in the test tube by tapping the bottom of the test tube on the desk or with your hand. Be careful to avoid generation of aerosol.
  3. Transfer a drop of the mixed sediment onto a clean glass slide and cover with a cover slip.
  4. Examine the prepared slide under the microscope using ×10 and ×40 objective lens. Make sure the condenser iris of the microscope is closed sufficiently in order to get a good contrast while viewing the fields of the slide

NOTE: Make sure that the contents of the centrifuge machine (test tubes) are balanced i.e. only even numbers of test tubes are to be allowed in the centrifuge, in order to avoid breakage of the centrifuge and subsequent damage to the centrifuge machine. Odd numbers of test tubes should not be allowed in the centrifuge before its operation.

REPORTING OF THE RESULT:

Report the appearance of the urine specimen examined. Presence of any of the following: WBC’s, RBC’s, casts, crystals, yeast cells, bacteria e.t.c. is reported using + sign depending on the number or amount found. Their absence is reported as “nil”.

Urine is H2O containing the H2O – soluble waste products removed from the blood stream through the kidney. Bacteriological examination of urine is done when the signs and symptoms of an infection or disease points to UTI’s, hypertension, and renal insufficiency. It should be done in persons with suspected systemic infection or fever of an unknown origin, and it is also desirable for women in the first trimester of pregnancy.

DEFINITIONS OF SOME TERMS ASSOCIATED WITH URINE ANALYSIS

Bacteriuria: This is the presence of bacteria in urine.

Cystitis: This is the infection of the urinary bladder.

Haematuria: This is the presence of blood in urine.

Pyuria: This is the presence of pus cells (dead WBC’s) in urine.

Pyelonephritis: This is the infection of the kidney.

Urethritis: This is the infection of the anterior urinary tract.

Dysuria: This is the pain experienced on passing urine.

Polyuria:  This is an increase in the volume of urine.

Oliguria: This is a decrease in the volume of urine.

Anuria: This is a cessation of urine flow.

Hypersthenuic urine: Urine with a high specific gravity.

Hyposthenuic urine: Urine with a low specific gravity.

Isosthenuic urine: Urine with a normal specific gravity.

URINE CHEMISTRY

AIM: To detect rapidly the abnormal levels of unorganized deposits/abnormal chemical constituents (ascorbic acid, bilirubin, blood, glucose, ketone, nitrite, pH value, protein, specific gravity, urobilinogen) in urine specimens as an aid in the diagnosis of urinary tract infections (UTI’s).

MATERIAL/APPARATUS: uncentrifuged urine specimen, combi – 9 – test strip, test tubes, test tube rack, discard jar.

METHOD/PROCEDURE:                                                       

  1. Describe the appearance of the urine specimen based on its colour and turbidity.
  2. Dispense the urine specimen into a clean test tube.
  3. Immerse the combi – 9 – test strip into the urine specimen and remove immediately, making sure that all the test area of the strip are covered.
  4. Hold the combi – 9 – test strip in a horizontal position upon removal from the urine specimen, in order to prevent interaction from adjacent test areas of the strip.
  5. Compare the reagent areas on the strip with that of a standard provided by the manufacturer of the test strip in order to read the result appropriately. This is normally done by holding the test           strip close to the colour chart on the container label of the test strip container. Ensure not to      contaminate the container with the urine on the strip while carrying out this procedure.

REPORTING OF THE RESULT:

Any deviation/change in colour of the test areas on the Strip when compared to the standard (which indicates normalcy) as provided by the manufacturer is indicative of an abnormality and should be reported as “positive” using the + sign depending on the extent of the change in colouration.

NOTE: The result of this test should not be read beyond 10 seconds, as a false positive result might be reported due to a drying effect.

SIGNIFICANCE OF THE UNORGANIZED DEPOSITS (ABNORMAL CHEMICAL CONSTITUENTS) DETECTED BY URINE TEST STRIP

Urine is produced and excreted by the kidneys. A normal human being excretes about 1 – 2 liters of urine every 24 hours (per day), though the volume of urine excreted by the kidney depends on our daily fluid intake, diet, climate (hotness & coldness of the environment), and other physiological factors of the body. The functional unit of the human kidney is called nephron and each of the kidneys contains over a million nephrons that controls and filters fluid that passes through the kidney.

pH: The normal reaction of freshly passed urine is slightly acidic, around pH 6.0. A normal urine contains about 95% of water, including electrolytes (magnesium, potassium, sodium, chloride, bicarbonate), and waste products of metabolism (urea, uric acid, and creatinine). The pH of the body is very important because it helps in maintaining a proper acid – base balance in the body.

Protein: An abnormal level of protein in urine is called proteinuria, and this condition can be caused by renal diseases such as pyelonephritis & glomerulonephritis, UTI’s, nephritic syndrome, and urinary schistosomiasis. Proteinuria should always be considered to indicate underlying disease until proved otherwise.

Glucose: Glucose can be found in the urine of diabetic patients, and occasionally in some healthy persons. Presence of more than the usual amount of glucose in urine is called glycosuria/glucosuria, and it is caused by a rise in the level of the blood glucose, and a reduced rate of reabsorption of glucose by the kidney tubules. Whenever glucose is found in urine, the blood glucose level should be measured in order to clear all doubts about diabetes.

Ketones: Ketonuria is a condition in which there are abnormal levels of ketone/ketone bodies in urine. Ketones are found in the urine of persons suffering from starvation, dehydration following prolonged vomiting and diarrhea, or untreated diabetes. Ketones are toxic to the brain and their accumulation can result to coma experienced by diabetic ketoacidosis patients.

Bilirubin: Bilirubin is found in the urine of persons with hepatocellular jaundice or cholestatic (obstructive) jaundice. It is not normally detected or found in the urine and when detected, the condition is called bilirubinuria.

Urobilinogen: Urine specimen is detected for the presence of urobilinogen when diagnosis points to abnormal haemolysis or liver disorders in which liver function is impaired. Nevertheless, it is normal to detect small amounts of urobilinogen in the urine.

Blood (Haemoglobin): The presence of free haemoglobin in urine is called haemoglobinuria. It occurs in bacterial infections like Escherichia coli septicaemia and typhoid fever, acute glomerulonephritis, UTI’s, severe falciparum malaria e.t.c.

Nitrite: The presence of nitrite in urine shows the presence of a bacterial infection as the urine of a healthy person does not contain nitrite. Detection of nitrate in urine is indicative of UTI’s which is normally caused by nitrate reducing bacteria. These pathogenic bacteria have the ability to reduce nitrate found in urine to nitrite

Specific Gravity: The relative mass density (formally known as specific gravity) of urine gives information regarding the concentrating and diluting ability of the kidneys. The normal relative mass density of urine varies from 1.002 – 1.025 depending on the state of hydration of the person and the time of the day. The ability of the kidneys to concentrate and dilute urine is reduced in renal failure of the kidneys.

 SEROLOGY

Serology is simply defined as the study of in vitro reaction between antigen and antibody. The word “In vitro” means “reaction occurring outside the body of a living organism”, that is in plate e.g. Petri dish plate. This reaction is opposed to in vivo reaction, which is a reaction that occur inside the body of a living organism. Most or majority of the test carried out in the microbiology laboratory are in – vitro test, in which the causative agent of a particular infection is deciphered by obtaining a specimen from a sick patient and testing it in the laboratory so that appropriate treatment plan can be organized or put in place for the patient experiencing the pathologic state.

In vivo test/experiments are usually employed in research laboratories, where animals are used for carrying out research. The serology unit of the clinical microbiology laboratory is solely responsible for testing patient’s serum and plasma in order to detect antibodies produced by the body in response to an infection. Serological techniques are usually used for the surveillance and early detection of diseases caused by microorganisms that are too difficult to culture in vitro.

Antibody is defined as a protein molecule synthesized/produced by the immune system on exposure of the body to an antigen. According to their physiological and biochemical properties, there are 5 different classes of antibodies produced by the human body: Immunoglobulin (Ig) A, D, G, E, and, M.

Antigen is defined as a foreign molecule/substance that can induce/start an immune response in our body. Typical examples of antigens are: various pathogens (bacteria, virus, protozoa, and fungi) that cause diseases in humans.

Every living organism especially man is constantly being attacked by one disease causing germs (pathogens) or the other in its lifetime. Man and other animals have evolved a strategy called “IMMUNE SYSTEM”, which they use to respond to these pathogens and evade their future threat to health. The immune system helps to free the body from microbe – burden. When an organism develops immunity (protection) to a specific pathogen after prior infection with same pathogen, the organism will remain free from that agent/pathogen for life.

This is the beautiful nature of the immune system which GOD blessed us with. Also very important is the segregative nature of our immune system. The immune system has the ability to different antigens (non – self) from the tissues, organs, and cells (self) in our body, so that the antibody it produces will only attack and fight the antigen/foreign substances that entered our body. Thus, antibodies are specific in their activities, binding only onto antigens. When this specificity of the immune system fails, an autoimmune disease/reaction occurs – in which the body starts fighting against itself.

The Rationale behind the use of serological tests in the laboratory diagnosis of an infection is that: when an infection occurs in the body of an organism, antibodies will be produced by the immune system against the antigen/pathogen responsible for the pathologic state of the patient. After the antibodies produced must have gone into action fighting and killing the antigen, they will die and remain in the body of the patient.

Now, to know whether such infection really took place, a preparation of the infecting pathogen which will serve as an antigen will be mixed or tested on a plate/test card with the serum/plasma obtained from the patient’s blood. A positive reaction shows that there was indeed an infection, while a negative reaction rules out the possibility of any infection. This is the basis for serological testing’s in the clinical microbiology laboratory, knowing the fact that both antigens and antibodies react specifically. Some of the serological tests used in the clinical microbiology laboratory will be the focus of the proceeding discussions. Enjoy yourself!

VENEREAL DISEASE RESEARCH LABORATORY (VDRL)

Venereal disease research laboratory (VDRL) test is the serological test used for the identification of Treponema pallidum, causative agent of syphilis infection in the clinical microbiology laboratory. Two types of serological test are available for the detection of T. pallidum antigens in the blood specimen of syphilis patients; and these are:

  1. Treponemal test.
  2. Non-treponemal test.

The treponemal test for serological diagnosis of syphilis infection uses a specific antigen to detect antibodies produced against T. pallidum in the serum of syphilis patients. Following the invasion of the body by T. pallidum, the immune system of the human host produces specific antibodies to counter the antigen. This serves as the basis for the treponemal test which uses a specific antigen to target specific antibodies produced in response to T. pallidum invasion of the body. However, this test cannot different between past infection and an active syphilis infection. The Treponema pallidum haemagglutination test (TPHA) and the fluorescent treponemal antibody absorption test (FTA-Abs) are the two available treponemal test for the serological detection of T. pallidum in the clinical microbiology laboratory.

Non-treponemal test include the VDRL and the rapid plasma regain (RPR) tests. Both VDRL and RPR test detect specific antibody-like substances (known as reagin) in the serum of syphilis patients. Reagin is an anti-lipioddal antibody made up of IgM and IgA. Cardiolipin-lecithin coated cholesterol particle (containing micro-particulate carbon/charcoal), a non-treponemal antigen is the antigen used for the VDRL and RPR tests. VDRL and RPR can be used to evaluate both active and past syphilis infections. However, VDRL and RPR tests are not always specific for the serological detection of T. pallidum because the reagin antibody can be present in other serum of people has other related diseases. Only the rapid plasma regain (RPR) test (which is the mostly used serological assay for syphilis diagnosis) which is more sensitive than the VDRL test, easier and faster to perform shall be expanded in this textbook.

Syphilis is a sexually transmitted disease (STD) that is caused by a spirochaete called Treponema pallidum. Natural infection with T. pallidum is only limited to the human host, and human infection is usually transmitted by sexual contact. Congenital syphilis in which the unborn fetus/infant of a pregnant mother is infected by T. pallidum can also occur. It is acquired in utero from the infected mother. Here, a pregnant mother that is syphilitic transmits T. pallidum to her unborn fetus via her placenta beginning in the 10th – 20th weeks of gestation. This can lead to miscarriages, stillborn, and even death.

RAPID PLASMA REGAIN (RPR) TEST

Aim: To detect the presence of reagin in the serum of patient’s blood as an aid in the serological diagnosis of syphilis infection.

PRINCIPLE: The principle is based on the immunological reaction between reagin antibodies and cardiolipin-cholesterol-lecithin antigen resulting to visible flocculation. The antigen suspension in the RPR test reagent contains charcoal-like particles which allows for the macroscopically visible flocculation on the test card or slide.

MATERIAL/APPARATUS: Blood specimen, VDRL antigen (i.e. cardiolipin-lecithin coated cholesterol particle), control reagents, test cards, centrifuge machine, Pasteur pipette, mixing stick or stirrer, timer.

METHOD/PROCEDURE:

  1. Bring the cardiolipin antigen reagent to room temperature (23oC – 29oC). This is done by bringing out the reagent from the refrigerator where it is stored when not in use, in order for it to attain room temperature before its usage. After use, the RPR test reagent should be stored in the refrigerator.
  2. Centrifuge the blood specimen in the centrifuge machine at 400 rpm for 5 minutes. This is done to sediment the red cell component of the blood specimen in order to obtain the serum component of the blood required to perform this test.
  3. Label the RPR test card with the laboratory number of the patient’s serum to be tested for pallidum infection. Note: Wells for positive (reactive), weakly positive and negative control sera (non-reactive) should also be labeled accordingly.
  4. Place a drop of the serum on the test card using the Pasteur’s pipette or disposable dropper. A new disposable dropper should be used for each sample.
  5. Shake the vial/phial containing the cardiolipin antigen reagent and add a drop of it to each of the test wells on the RPR test card including the test serum well.
  6. Mix the mixture together using a stirrer, mixer or the paddle end of the Pasteur’s pipette.
  7. Rock /rotate the test card for about 8 minutes after mixing. An automatic rotating machine could also be used to rotate the RPR test card if available.
  8. Observe the mixture macroscopically after the rocking, and look out for visible flocculation.

REPORTING OF THE RESULT:

Presence of flocculation after the 8 minutes implies that the patient’s specimen contains the reagin antibody, and this is reported as reactive because it is indicative of T. pallidum infection. While absence of flocculation indicates that the patient’s serum does not contain reagin antibodies, and this is reported as non – reactive.

NOTE: RPR test is a non – specific test used in the laboratory diagnosis of syphilis infection. It is only a screening test, and should be confirmed by carrying out other confirmatory test whenever the RPR test proves reactive. The RPR test could also give false positive result in the case of similar infections or reacting antibodies, but this barely occurs as opposed to the TPHA test where false positive result is more common.

WIDAL TEST

AIM: To detect the presence of agglutinins (O and H antibodies) in patient’s serum as an aid in the diagnosis of typhoid and paratyphoid fevers.

PRINCIPLE: The principle is based on the immunological reaction between agglutinins in patient’s serum and febrile antigen suspensions, resulting in visible agglutination within 60 seconds.

MATERIAL/APPARATUS: Blood specimen, centrifuge machine, Pasteur’s pipette, test card, timer, mixing stick, Salmonella typhi O and H antigens, S. paratyphi AO and AH antigens, S. paratyphi BO and BH antigens, S. paratyphi CO and CH antigens.

O = Somatic cell wall antigen (blue in colour).

H = Flagella antigen (red colour).

“The letters A, B, & C” are used to differentiate the 3 strains of S. paratyphi.

METHOD/PROCEDURE: 

  1. Bring the test reagents to room temperature (23oC – 29oC).
  2. Centrifuge the blood specimen in the centrifuge machine at 400rpm for 5 minutes, in order to sediment the red cell component of the blood and thus obtain the serum component of the blood specimen required to undertake this test.
  3. Using the Pasteur’s pipette, place a drop of the serum on 8 different spots of the test card.
  4. Shake the vials containing the antigen reagents and place a drop of the Salmonella typhi O and H antigens, paratyphi AO and AH antigens, S. paratyphi BO and BH antigens, and S. paratyphi CO and CH antigens respectively on the 8 drops of the serum on the test card.
  5. Mix the mixture together using a mixer or the paddle end of the Pasteur’s pipette.
  6. Rock and rotate the test card for 60 seconds.
  7. Observe the test card immediately after rocking, for visible agglutination.

REPORTING OF THE RESULT:

The Widal test is reported by giving the titer values for both the O and H antibodies. This is done by observing any degree of agglutination that is visible within 60 seconds. The highest dilution of serum in which agglutination occurs is taken as the antibody titer. The result is reported using titer values like: 1:20, 1:80, 1:160, 1:30 e.t.c. depending on the degree of visible agglutination observed macroscopically. Titer values less than 80 (e.g. <20) are considered non – reactive or negative, while those above 80 are considered significant. The Widal test result should not be read beyond the 60 seconds time interval, because of the possibility of drying effect which might give a false positive result.

Example of a Widal test result report is thus:

O                 H

Salmonella typhi                                                                                       <20             <20

Salmonella paratyphi A                                                                           <20             <20

Salmonella paratyphi B                                                                           <20             <20

Salmonella paratyphi C                                                                           <20             <20

Significant titer: >80.

Widal test (pronounced as’ Vidal’) is a serological technique which is used to test for the presence of Salmonella antibodies (O and H antibodies) in a patient’s serum. Though good for the investigation of typhoid and paratyphoid fevers caused by S. typhi and S. paratyphi, it is of no value in the investigation of food poisoning caused by S. typhimurium. This test can also be performed by a tube method, as only the slide method is expanded here.

Agglutination is defined as the clumping of blood cells. It is the reaction between a particular antigen and an antibody, which results to the formation of a visible clump.

The ‘O’ antigen is the somatic cell wall of the infecting organism (Salmonella). It is the first to be produced during an infection, and an increase in the titer value of ‘O’ during testing is indicative of the presence of an infection. The ‘O’ antigen suspension used in this test is bleu in colour.

The ‘H’ antigen is the flagella antigen. Its increase in titer value during testing is often not significant as it shows past or old infection. It is also not considered as it can suggest prior immunization of the patient against the infection. The ‘H’ antigen suspension used in this test is red in colour. The difference in colouration helps to differentiate the 2 antigen preparations in order to prevent chaos in reporting the test result.

Salmonella are a group of bacteria in the family Enterobacteriaceae. They cause different forms of infections in humans including: enteric fevers, enterocolitis, and bacteremia. The pathogen gain entrance into the human body via the oral route through contaminated food and water. They can easily spread to susceptible hosts via a direct contact with carriers or infected people, and the best and most adequate way of preventing their spread and contamination is by observing a good personal hygiene. 

ANTI – STREPTOLYSIN O (ASO) TEST 

There are two types of serological tests that are available for the detection of antibodies produced against an invading Streptococcal pathogen.

  1. ASO latex agglutination test – This test detects raised ASO titers (200 IU or higher) in the serum of infected patients. IU = international unit.
  2. ASO tube test – This is usually used as a confirmatory test to validate positive results of the ASO latex agglutination test. Titers less than 50 IU is indicative of a negative test result.  

AIM: To detect the presence of anti – streptolysin o (ASO) antibodies in the serum of patient’s as an aid in the diagnosis of streptococcal infections e.g. pharyngitis.

PRINCIPLE: The principle is based on the immunological reaction between ASO antibodies with Streptolysin O antigen coated onto latex particles, to form visible agglutination.

MATERIAL/APPARATUS: Blood specimen, ASO antigen reagent (a suspension of latex particles coated with Streptolysin O), centrifuge machine, timer, Pasteur’s pipette, test card, mixing stick.

METHOD/PROCEDURE:

  1. Bring the ASO antigen test reagent to room temperature (23oC – 29oC).
  2. Centrifuge the blood specimen in the centrifuge machine at 400rpm for 5 minutes, in order to sediment the red cell component of the blood and obtain the serum.
  3. Place a drop of the serum onto the test card using the Pasteur’s pipette.
  4. Shake the vial containing the ASO antigen test reagent very well, and place a drop of the reagent onto the serum on the test card.
  5. Mix the mixture together using the mixing stick or the paddle end of the Pasteur’s pipette.
  6. Rock/rotate the test card for 2 minutes.
  7. Observe macroscopically, after the time interval for visible agglutination.

REPORTING OF THE RESULT:

Presence of agglutination within the 2 minutes of rocking indicates that the patient’s serum contains ASO antibodies. This is a positive result and is reported as thus: Concentration of ASO > 200 IU/ml.

Absence of agglutination within the 2 minutes of rocking indicates that the patient’s serum does not contain ASO antibodies that are up to 200 IU/ml. This is a negative result and is reported as thus: Concentration of ASO < 200 IU/ml.

IU/ml = International Unit per mil

Streptococcus pyogenes is the main human pathogen associated with local or systemic invasion, and post streptococcal immunologic disorders in the body. They are β – haemolytic and contain the group A antigen which helps in classifying them as “the β – haemolytic group A S. pyogenes” under the Lancefield classification of Streptococci. They have the exceptional ability to hemolyze red blood cells (RBC’s) completely with the release of haemoglobin in vitro, and are thus called β – haemolytic Streptococcus while those that only hemolyze RBC’s incompletely are called α – haemolytic Streptococcus.

Streptococcus pyogenes produces 2 different types of hemolysins (streptolysins) that help it to hydrolyze/breakdown RBC’s, viz: Streptolysin O and Streptolysin S. The former is antigenic while the latter is not. That is, streptolysin ‘O’ stimulates the production of antibody by the body while streptolysin ‘S’ does not stimulate the production of antibody by the body following an infection. This antibody that is produced by the body following an infection with S. pyogenes is called anti – streptolysin O (ASO) antibody.

Anti – streptolysin O (ASO) antibody is produced by the human body in response to infection with S. pyogenes and other Streptococci that produce streptolysin ’O’. ASO combines quantitatively with streptolysin ‘O’ produced by S. pyogenes in vivo. ASO blocks hemolysis by streptolysin ‘O’. This phenomenon forms the basis of a quantitative test for the antibody (ASO). An ASO serum titer in excess of 160 – 200 units is considered abnormally high and suggests either recent infection with Streptococci or persistently high antibody levels due to an exaggerated immune response to an earlier exposure in a hypersensitive person. 

RHEUMATOID FACTOR (RF) TEST

AIM: To detect the presence of rheumatoid factor autoantibody in the serum of patient’s as an aid in the diagnosis of rheumatoid arthritis.

PRINCIPLE:  The principle is based on the immunologic reaction between the rheumatoid factor autoantibody in patient’s serum with the corresponding IgG coated onto latex particles, resulting in visible agglutination.

MATERIAL/APPARATUS: Blood specimen, centrifuge machine, timer, test card, Pasteur’s pipette, rheumatoid factor latex reagent (a component of latex particles, commercially prepared IgG, & 0.1% sodium azide serving as a preservative).

METHOD/PROCEDURE: 

  1. Bring the rheumatoid factor test reagent to room temperature (23oC – 29oC).
  2. Centrifuge the blood specimen at 400rpm for 5 minutes in the centrifuge machine in order to sediment the red cell component of the blood, and obtain the serum required for this investigation.
  3. Place a drop of the serum on a test card using the Pasteur’s pipette.
  4. Shake the vial containing the rheumatoid factor test reagent, and place a drop of it on to the serum on the test card.
  5. Mix the mixture together using a mixing stick or the paddle end of the Pasteur’s pipette.
  6. Rock/rotate the test card for about 3 minutes.
  7. Observe the test card macroscopically for any visible agglutination within the time interval.

REPORTING OF THE RESULT:

Presence of agglutination indicates that the patient’s serum contains rheumatoid factor autoantibodies, and is thus reported as positive. Absence of agglutination is a negative result, and it shows that the patient’s serum does not contain rheumatoid factor autoantibodies.

Rheumatoid arthritis is a chronic systemic inflammatory autoimmune disease generally characterized by swelling and pain in the joints of our body. Its onset is characterized by the detection of rheumatoid factors in the serum of patient’s whole blood, who suffer from rheumatoid arthritis.

Autoantibody is an antibody produced by the body, and directed against normal body tissue. It results when autoimmunity sets in. Autoimmunity is the process by which the body mounts an immune response against a normal body component. This results to autoimmune diseases (e.g. rheumatoid arthritis) which are diseases that emanate when the body’s immune system fails to recognize and differentiate self from non – self (pathogens). Rheumatoid arthritis is organ specific, and it is most common in women and afflicts them between 40 & 60 years of age.

Rheumatoid factors are autoantibodies produced in the body of rheumatoid arthritis patients. It is an IgM class of antibody. It is reactive at the crystallizable fragment (Fc) region of IgG, and this forms the basis for this test. The rheumatoid factor autoantibody reacts with IgG to form IgM – IgG complex which deposits in the joints together with other mediators of the immune system e.g. plasma cell, neutrophils e.t.c. These complexes activate complement reaction leading to a type hypersensitivity reaction. This finally results to the chronic inflammation of the joints.

Serum (Sera – plural) is the yellowish/golden fluid that remains after blood coagulates, and the red cells have formed clot. It is plasma minus the clotting factor fibrinogen. Serum contains mainly antibodies produced during the pathologic state of a patient, and that is why serum is often required to perform most serological tests carried out in the clinical microbiology laboratory.

Plasma is the red blood cell free, fluid portion of blood which contains all the clotting factors of blood. It consist of H2O containing a large number of dissolved substances, including proteins, salts (Na, K, Chlorides, & bicarbonates), food materials (glucose, fats, amino acids), hormones, vitamins, & excretory materials. Plasma is obtained from a whole blood by placing/collecting the blood specimen in an anticoagulant bottle i.e. bottles that contains substances that prevent blood from clotting. Examples of anticoagulants are: ethylene diamine tetra – acetic acid and heparin. The above serological tests explained in this chapter can be performed using plasma, with the exception of rheumatoid factor test which can only be performed with serum because plasma contains fibrinogen (a blood clotting factor) which may cause non – specific agglutination of the latex particles contained in the rheumatoid factor test reagent. This gives a false positive result.

MYCOLOGY

Mycology is defined as the study fungi. The mycology unit of the clinical microbiology laboratory is largely responsible for handling, examining, and processing of patient’s specimens suspected to contain fungal organisms. Our concern here is on fungi that are of medical importance i.e. fungi that cause infections in humans. Fungi reside in nature, with the soil accounting for a large amount of their dwelling place. They play important role in our ecosystem, such as breaking down of organic matter and recycling of organic matter in the environment, thus getting rid of organic wastes from the environment. Their saprophytic nature has gained them this advantage over other group/category of microorganisms. Fungi like some bacteria have proved worthy in the manufacture/production of a wide variety of antibiotics which are used today in clinical medicine to treat infectious disease caused by microbes/pathogens. Example is the fungus Penicillium notatum from which the first antibiotic (penicillin) was produced from in late 1920’s.

Fungi unlike bacteria are eukaryotes/eukaryotic organisms and they grow in two basic forms viz: the mould form and the yeast form. The moulds grow by the production of multicellular filamentous colonies which consists of branching cylindrical tubules known as hyphae. Rhizopus nigricans is an example of a mould.  Yeasts are single celled, and they are usually spherical to ellipsoid in shape with varying diameters. Most yeasts reproduce by budding. Saccharomyces cerevisiae is a typical example of yeast.

Fungal infections are called mycoses. Candidiasis caused by Candida albicans, and dermatophytosis caused by dermatophytes like Trichophyton spp and other species of Candida are the common mycoses with the highest incidence in humans. These mycoses are caused by fungi that are part of the body’s normal microbial flora, fungi that are highly adapted to survival on the human body. Fungal infections (mycoses) are classified as: Superficial, Systemic, Opportunistic, Cutaneous, and Subcutaneous mycoses depending on the part of the body that is infected and the site via which they gain entrance into their human host.

Fungi are usually larger than bacteria, and most occur in nature and grow readily on simple sources of nitrogen and carbohydrates. Sabouraud’s dextrose agar (SDA) which contains glucose and modified peptone has been used to grow fungi because it does not readily support the growth of bacteria. Antibiotics can also be added to SDA during its preparation, when culturing medical fungi from non-sterile patient’s specimens. The essence of adding antibiotics is to prevent/inhibit the growth of bacteria and saprophytic moulds that might affect fungal growth. Gentamicin & chloramphenicol are used for the bacteria while cycloheximide is used against the saprophytic mould.

Two other very important reagents/stains which aid and help the easy identification of fungal organisms in the mycology unit are: potassium hydroxide (KOH) and lactophenol cotton blue.

Potassium hydroxide (KOH) is used to the KOH mount in the mycology unit. Fungal specimens (skin scrapings, hair, or nails) are softened and cleared first in KOH solution prior to their direct microscopy. The KOH helps to digest the keratinized tissues (portions) surrounding the specimen so that the fungal spores (conidia) and hyphae can be exposed and seen under the microscope.

Lactophenol cotton blue is used to mount fungal cultures in the mycology unit. It helps in giving good contrast that is required during viewing with the microscope so that fungal structures can be easily seen and identified.

Specimens encountered in the mycology unit include:

  • Skin scrapings
  • Cornea clippings
  • Hair
  • Nail clippings
  • Collection of specimens that cannot be scraped, using sterile swab stick
  • Other specimens handled here are biopsy, sputum, aspirate, pleural fluid, pus & CSF

Collection of fungal specimens in the mycology unit

Fungal specimens unlike other specimens encountered in the clinical microbiology laboratory are collected most often without necessarily asking the patient to do it. Adequate precautions are also observed because of the ease with which fungal elements can be easily aerosolized.

Some of the specimens and how they are collected are as follows:

  1. Cover your face with face mask and put on hand gloves before proceeding with the collection.
  2. Cleanse the affected area with 70% ethanol.
  3. Collect skin scales, crusts, pieces of nails or hairs on a clean white piece of paper that is about 5 cm square.
  4. Skin scales: Collect skin scales by scraping the surface of the margin of the lesion using a sterile scalpel blade. Make sure not to bleed the patient.
  5. Crusts: Crusts are collected by removing part of the crust nearest to healthy skin using sterile scissors and tweezers.
  6. Nail pieces: Nail pieces are collected by a procedure called nail clipping. Sniping of the infected part of the nail are collected using sterile scalpel blade or scissors. Collect sniping from beneath the nail in cases where the nail is thickened.
  7. Hairs: Hairs are collected by removing dull broken hairs from the margin of the lesion using a sterile tweezers or by scraping the scalp of the head with a scalpel blade.
  8. Finally, fold the paper onto which the specimen was scraped or collected, in order to enclose the specimen and protect it from dispersal. Label the paper with the patient’s data and proceed for mycological investigation.

Note: In the collection of clinical specimens for fungal investigation, it is very important to avoid any form of bleeding so that the specimen is not contaminated with blood. Wash hands with detergents immediately after the collection process. Also, the patient is informed to refrain from any sort of medication 3 days prior to collection of the specimen.

 MICROSCOPY/KOH MOUNT OF FUNGAL SPECIMEN

AIM: To digest and look out for fungal elements (hyphae & spores) in fungal specimens prior to their culturing.

MATERIAL/APPARATUS: Fungal specimen, face mask, 10% KOH, microscope, glass slide, cover slip, scalpel blade, inoculating loop, 70% ethanol, Bunsen burner, clean white paper, cotton wool, hand gloves.

METHOD/PROCEDURE

  1. Cover your face with a face mask in order to avoid inhalation of fungal spores during specimen collection (scraping).
  2. Clean the affected area from which the specimen will be collected on the patient’s body with 70% ethanol and allow to dry.
  3. Scrape the affected area onto a clean with paper using a sterile scalpel blade. Make sure not to bleed the patient while doing this, and that the patient’s data are written on the paper prior to the scrapping.
  4. Disinfect the site of collection with 70 % ethanol after the scrapping. This will help to prevent the spread of fungal elements.
  5. Place a drop of the 10% KOH solution on a clean glass slide.
  6. Transfer a small portion/piece of the specimen to be tested, onto the KOH solution using a sterile inoculating loop.
  7. Emulsify the preparation using the inoculating loop, and cover with a cover slip.
  8. Place the prepared slide on an undisturbed surface for about 24 hrs before viewing with the microscope. The time lap is to allow for the KOH solution to act on the specimen, and further enhance the clearing of the keratinized portion of the specimen so that the fungal elements present can be exposed.
  9. View the slide under the microscope using the ×10 and ×40 objective lens.

REPORTING OF THE RESULT:

Look out for the presence of any fungal element (spores & hyphae) in the preparation. Make sure that you examine several fields of the slide before making your final report, so as to clear all doubts. Presence of any fungal element is reported while their absence is reported as thus: No fungal elements seen. 

CULTURING OF FUNGAL SPECIMENS 

AIM: To isolate and identify pathogenic fungi in patient’s specimens as an aid in the diagnosis of fungal infections (mycoses)

MATERIAL/APPARATUS: Patient’s specimen, incubator, inoculating loop, Bunsen burner, S+C tube, S+C+A tube, lactophenol cotton blue reagent,  glass slide, cover slip, microscope.

S = Sabouraud’s dextrose agar (SDA)

C = Chloramphenicol

A = Actidine (cyclohexamide)

METHOD/PROCEDURE:

  1. Label the S+C and S+C+A tubes with the patient’s biodata (e.g. name, date of culturing, laboratory number, and type of specimen).
  2. Collect the specimen using a sterilized inoculating loop.
  3. Inoculate the specimen on the slope and butt of the S+C and S+C+A tubes.
  4. Incubate the tubes in the incubator at 28oC for about 21 days.
  5. Check the incubated tubes for fungal growth at different time intervals before the 21 days elapses.
  6. After the 21 days, bring out the S+C and S+C+A tubes from the incubator.
  7. Make wet preparations of the fungal growths in both tubes on a clean glass slide using the lactophenol cotton blue stain.
  8. Cover the prepared slide with a cover slip and view with the ×10 and ×40 objective lens of the microscope.

REPORTING OF THE RESULT:

The name of the fungus responsible for the disease condition of the patient is reported after a thorough microscopical investigation is carried out. Familiarity with fungal elements and structures also helps in this process.

GERM TUBE TEST

AIM: To identify Candida albicans from fungal cultures as an aid in the diagnosis of Candidiasis.

MATERIAL/APPARATUS: Fungal culture, glass slide, cover slip, microscope, lactophenol cotton blue, Bunsen burner, inoculating loop, water bath or incubator, micro – pipette, test tube, test tube rack, horse serum or human serum, Pasteur pipette.

METHOD/PROCEDURE:

  1. Using a micro – pipette, pipette or transfer 0.5 ml (500 µl) of human/horse serum into a sterile test tube.
  2. Inoculate a portion of the fungal culture into the serum in the test tube using a sterile inoculating loop.
  3. Place the test tube containing both the fungal culture and the serum in a water bath or incubator at 35 – 37oC for about 2 – 3 hours.
  4. Transfer a drop of the serum – fungal culture to a glass slide using a Pasteur’s pipette.
  5. Cover the preparation with a cover slip.
  6. Examine the prepared slide under the microscope using the ×10 and ×40 objective lens. Make sure that the condenser iris of the microscope is sufficiently closed in order to get a good contrast. A drop of lactophenol cotton blue stain can also be added to the preparation if desired in order to stain the yeast cells for a proper/clear view.

REPORTING OF THE RESULT:

Look out for germ tubes i.e. sprouting/growing yeast cells that have tube – like outgrowth. If found, report the culture as: Candida albicans isolated.   But if these sprouting yeast cells are not seen, report the culture as: yeast other than C. albicans isolated.

NOTE: Only C. albicans and C. dubliniensis (a rarer oral pathogen) can produce germ tubes under the above described conditions. Other species of the genius Candida are germ tube negative under the above conditions, and thus require further tests to identify them.

Candida albicans is yeast which falls under the genus of fungi known as Candida. Several species of Candida are capable of causing candidiasis in humans. Candida species are members of the normal flora of the skin, mucous membranes, and gastrointestinal tract of humans. The indiscriminate administration of antibiotics in humans can further increase the amount of Candida in our intestinal tract, and they can enter the circulation by crossing intestinal mucosa, and become systemic. Candida vaginitis is a common infection experienced by women during pregnancy. Candida infections are opportunistic mycoses, occurring in debilitated people i.e. persons whose immune systems are weak or compromised as a result of a prior infection or something else like stress. Candida infections of the mouth and oesophagus are most common in people who have HIV infections.  The infection can be treated by the administration of antifungal drugs like amphotericin B, nystatin or fluconazole. Avoiding disturbance to the normal balance of microbial flora and host defenses of the body is the most important preventive measure for candidiasis.

REFERENCES

Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. 2nd Cambridge University Press, UK. Pp. 178-187.

Willey J.M, Sherwood L.M and Woolverton C.J (2008). Harley and Klein’s Microbiology. 7th ed. McGraw-Hill Higher Education, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

World Health Organization (1993). Laboratory Biosafety Manual, 2nd edn. Geneva: WHO.

World Health Organization (2003). Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens.  WHO/EMC/97.3. Geneva: WHO.

Stevens, Christine Dorresteyn (2010). Clinical immunology and serology. Third edition. F.A. Davis Company, Philadelphia.

Summers W.C (2000). History of microbiology. In Encyclopedia of microbiology, vol. 2, J. Lederberg, editor, 677–97. San Diego: Academic Press.

Roberta B. Carey, Mindy G. Schuster and Karin L. McGowan (2008). Medical microbiology for the new curriculum: a case based approach. John Wiley & Sons, Inc, Hoboken, New Jersey, USA.

Roberts L, Janovy J (Jr) and Nadler S (2012). Foundations of Parasitology. Ninth edition. McGraw-Hill Publishers, USA.

Ryan K, Ray C.G, Ahmed N, Drew W.L and Plorde J (2010). Sherris Medical Microbiology. Fifth edition. McGraw-Hill Publishers, USA.

Shea Y.R (1992). Specimen collection and transport. Section 1. Aerobic bacteriology, p.111-1310. In H.D. Isenberg (ed. In Chief), Clinical Microbiology Procedures Handbook. American Society of Microbiology, Washington, D.C.

Mandell G.L., Bennett J.E and Dolin R (2000). Principles and practice of infectious diseases, 5th edition. New York: Churchill Livingstone.

Mandell GL, Bennett JE and Dolin R (2004). Principles and Practice of Infectious Diseases. Volumes 1 & 2. Sixth Edition. Churchill Livingstone, New York.

Maza Luis dela, Pezzlo M.T, Shigei J.T, Tan G.L and Peterson Ellena M (2013). Colour Atlas of Medical Bacteriology. Second edition. American Society of Microbiology Press, USA.

Miller J.M (1999). A Guide to Specimen Management in Clinical Microbiology, 2nd ed. ASM Press, Washington D.C. USA.

Murray P.R, Baron E.J, Jorgensen J.H., Pfaller M.A and Yolken R.H (2003). Manual of Clinical Microbiology. 8th edition. Volume 1. American Society of Microbiology (ASM) Press, Washington, D.C, U.S.A.

Murray P.R, Baron E.J, Jorgensen J.H., Pfaller M.A and Yolken R.H (2003). Manual of Clinical Microbiology. 8th edition. Volume 2. American Society of Microbiology (ASM) Press, Washington, D.C, U.S.A.

Leave a Reply

Your email address will not be published. Required fields are marked *