Category Archives: LABORATORY PROCEDURES

HOW TO PREPARE CULTURE MEDIA

● Prepare media made from dehydrated products in as damp-free an environment as possible. To prevent the risk of inhaling fine particles of dehydrated media, wear a dust mask while handling dehydrated media.

● Wash the hands immediately after preparing media.

● Once the ingredients are weighed, do not delay in making up the medium. Follow exactly the manufacturer’s instructions.

● Use completely clean glassware, plastic or stainless steel equipment that has been rinsed in pure water. The container in which the medium is prepared should have a capacity of at least twice the volume of the medium being prepared.

● Use distilled water from a glass still. Deionized water can also be used providing the exchange resins do not contain substances inhibitory to bacteria (preparation of deionized and distilled water in district laboratories is described in Water containing chlorine, lead, copper, or detergents must not be used. Besides containing substances harmful to bacteria, impure water can alter the pH of a medium or cause a precipitate to form.

● Add the powdered or granular ingredients to the water and stir to dissolve. Do not shake a medium but mix by stirring or by rotating the container.

● When heating is required to dissolve the
medium, stir while heating and control the heat to prevent boiling and foaming which can be dangerous and damage the medium, e.g. DCA or TCBS agar. Overheating a medium can alter its nutritional and gelling properties, and also its pH.

● Autoclave a medium only when the ingredients are completely dissolved. Always autoclave at the correct temperature and for the time specified

● Dispense medium in bottles or tubes in amounts convenient for use. Know the length of time prepared media can be stored without deteriorating (take into account storage temperature)

DIFFERENT TYPES OF CULTURE MEDIA


For a culture medium to be successful in growing the pathogen sought it must provide all essential nutrients, ions, and moisture, maintain the correct pH and osmotic pressure, and neutralize any toxic materials produced. It is also essential to incubate the inoculated medium in the correct atmosphere, at the optimum temperature and for an adequate
period.

The main types of culture media are:
● Basic
● Enriched
● Selective
● Indicator
● Transport
● Identification

BASIC MEDIA

These are simple media such as nutrient agar and nutrient broth that will support the growth of microorganisms that do not have special nutritional requirements.
They are often used in the preparation of enriched media, to maintain stock cultures of control strains of bacteria, and for subculturing pathogens from differential or selective media prior to performing biochemical and serological identification tests.

ENRICHED MEDIA

Enriched media are required for the growth of organisms with exacting growth requirements such as
H. influenzae, Neisseria species, and some Streptococcus species. Basic media may be enriched with whole or lyzed blood, serum, peptones, yeast extract, vitamins and other
growth factors. An enriched medium increases the numbers of a pathogen by containing all the necessary ingredients to promote its growth. Such a medium is often used for specimens collected from sites which are normally sterile to ensure
the rapid multiplication of a pathogen which may be present only in small numbers.

ENRICHMENT MEDIA

Enrichment media: This term is usually applied to fluid selective media which contain substances that inhibit the growth of unwanted organisms,e.g. Rappaport-Vassiliadis broth which is often used as an enrichment medium for Salmonella serovars in faeces.

SELECTIVE MEDIA

These are solid media which contain substances (e.g. bile salts or other chemicals, dyes, antibiotics)
which inhibit the growth of one organism to allow the growth of another to be more clearly demonstrated. A selective
medium is used when culturing a specimen from a site having a normal microbial flora to prevent unwanted contaminants
overgrowing a pathogen. Media made selective by incorporating antibiotics are usually expensive.

Other ways to select organisms:
Incubation conditions may be used to select organisms, e.g. P. aeruginosa is inhibited by anaerobic conditions. Also the
pH of a medium may make it selective for a particular
organism, e.g. V. cholerae can be isolated on an alkaline medium such as TCBS agar. Temperature may also help to
select an organism e.g. Listeria monocytogenes can grow at 4° C whereas other organisms are inhibited. Growth, however, is slow.

INDICATOR MEDIA

These are media to which dyes or other substances are added to differentiate microorganisms. Many differential media distinguish between bacteria by incorporating an indicator which changes colour when acid is produced following fermentation of a specific
carbohydrate e.g. MacConkey agar.

Many media used to isolate pathogens are both selective and enrichment or both selective and differential.

TRANSPORT MEDIA

These are mostly semisolid media that contain ingredients to prevent the overgrowth of commensals and ensure the survival of aerobic and anaerobic pathogens when specimens cannot be cultured immediately after collection. Their use is particularly important when transporting microbiological specimens from health centres to the district microbiology laboratory or specimens to the Regional Public Health Laboratory.

Examples of transport media include
Cary-Blair medium for preserving enteric pathogens and Amies transport medium for ensuring the viability of gonococci.

IDENTIFICATION MEDIA

These include media to which substrates or chemicals are added to help identify bacteria isolated on primary cultures. Examples include peptone water sugars, urea broth, and Kligler iron agar. Organisms are mainly identified by a change in the colour of the medium and or the production of gas. Organisms used to inoculate identification media must be first isolated in pure culture.

USING INDIAN INK FOR FUNGAL DETECTION

This procedure is employed to observe capsule around the yeasts especially Cryptococcus neoformans in CSF. Some bacteria with capsule eg klebsiella stain with Indian ink but are one fourth the size of yeast and are not budding.

Indian ink is used as definitive evidence of Cryptococcus neoformans but other studies such as dark brown pigment production on caffeic acid agar, direct antigen testing are also used.

1. Place a small drop of India ink on a grease free slide containing the test sample

Process different specimens as follows:

2. Fluid samples like CSF should be centrifuged and the sediment used on the slide.

3. In thick specimens like pus and sputum add drop of 10% KOH along with India ink to improve visualisation.

4. Leukocytes and tissue cells can be dissolved by adding a drop of 10% KOH.

5. Observe under ×10 and confirm by ×40

6. Record the findings

HOW CAN WE ESTIMATE URINARY CHLORIDE


1. Pipette 1 ml of clear urine into a test tube of the same size as the small standard tubes. If the urine is cloudy, filter or centrifuge it to obtain a clear sample for testing.

2. Add 1 ml of acidified silver nitrate reagent to the urine and mix. Caution: The reagent is corrosive. Also handle it
with care because although colourless it will stain skin, clothes, bench surfaces, etc, and the brown colour is difficult to remove.

3. Examine the urine for a cloudiness. Estimate the approximate concentration of chloride in the urine by matching the cloudiness with the standards. Matching is best carried out against a dark background.

4. Report the approximate chloride concentration in the urine in mmol/l.
If the urine remains clear after adding the reagent report the test as ‘no chloride detected’. Use a ‘normal’ urine as a positive control.

GRAM STAINING PROCEDURE FOR FUNGAL DETECTION

Stains walls containing protein sugar complexes called peptidoglycan. Typically have low lipid content. Gram positive fungi stain violet. Gram negative fungi decolourise and stain pink with safranin or neutral red counter stain.

1. Label a clean grease free slide with sample identification number.

2. Place a drop of normasaline and emulsify a small sample to make a thin smear

4. Apply crystal violet stain to the smear for 60 seconds. Drain stain and wash with water.

3. Heat-fix the smear.

5. Add Gram’s Iodine for 60 seconds and the drain excess.

6. Decolourise with acid alcohol or Acetone for 30 seconds.

7. Counterstain with safranin or neutral red for 60 seconds and then drain excess.

8. Rinse and allow to air dry.

9. Apply immersion oil
Examine under microscope at X100.
Record the findings

LACTOPHENOL COTTON BLUE (LPCB) FOR FUNGAL DETECTION

Used for visualisation of fungal elements and structures. Mainly performed from fungal colonies and used for identification.
The phenol in the stain will kill any organisms while the lactic acid preserves fungal structures. Cotton blue stains the chitin in fungal cell walls. The same structures may be seen as with wet mount; however LPCB preparation can be made permanent.

How to prepare the LPCB stain


Concentrated phenol………………………20 mls
Lactic acid………………………………….20 mls
Glycerol………………………………………40 mls
Cotton blue(china blue)………………… …0.05g
Distilled water…………………………..20 mls
Dissolve cotton blue in distilled water, then add the rest of the ingredients. Mix well.

Procedure of Using the LFCB stain

1. Place 1 drop of LPCB on clean grease free slide.

2. Immerse fungal colonies with help of swab or Scotch tape or tease mount.

3. Apply coverslip gently.

4. Visualize under microscope at X10 then X40.

5. Record the findings

SALINE MOUNT FOR FUNGAL DETECTION

Fungal elements that may be observed are budding yeast, hyphae and pseudohyphae, conidia, thin branching filaments resembling bacteria(fungus like bacteria),granules ,or coccidioides immitis spherules.

Preparation of reagent

Normal saline:
Nacl 0.85 g in 100 mls distilled water.


The procedure of saline mount

1. Place one drop of specimen on a glass slide and add one drop of saline.

2. Place coverslip and observed under low and high power under microscope.

Organism appear refractile, shinny or slightly green. Differentiate yeast cells from RBCs; yeast contain inclusion while others do not.

Look for spores,branching hyphae

See also:

10% KOH MOUNT

USING 10% KOH FOR FUNGAL DETECTION

If the specimen is skin, hair or nail, the background cellular material may mask fungal elements. Potassium hydroxide will dissolve the keratin in these specimens, thus making any fungi more visible.

If there are a lot of cellular material in sputum or vaginal secretions, KOH mount may be prepared to dissolve the background thus making any yeast more visible.

However a saline mount must be additionally done to quantitate epithelial cells and WBCs and note presence of Trichomonas.

How to prepare 10% KOH

Potassium hydroxide……….…………10 g
Glycerol…………………………….20 mls
Distilled water………………………80 mls

Dissolve KOH in water, and then add glycerol. Glycerol prevents crystallization of the reagent and allows KOH preparations to be maintained for two days before drying up.

The procedure of using 10% KOH


1. Place specimen on clean grease free slide.

2. Add a drop of 10% KOH.

3. Apply coverslip and gently warm the slide over a flame.

4. Gently press the coverslip to spread the already softened tissue evenly on the slide.

5. Examine under a microscope X10 then X40.

6. Record the findings

Potassium hydroxide is a highly corrosive deliquescent chemical, therefore handle it with great care and make sure the stock bottle of chemical is tightly stoppered after use’.

SOURCES OF ERROR WHEN MEASURING HAEMOGLOBIN PHOTOMETRICALLY

● Not measuring the correct volume of blood due to poor technique or using a wet or chipped pipette.

● When using anticoagulated venous blood, not mixing the sample sufficiently.

● Not ensuring that the optical surfaces of a
cuvette are clean and dry and there are no air bubbles in the solution.

● Not protecting a colorimeter or haemoglobin meter from direct sunlight and not checking the performance of an instrument or maintaining it
as instructed by the manufacturer. A common error when using a filter colorimeter is using a glass filter which is not clean.

● Not checking a diluting fluid such as Drabkin’s for signs of deterioration as explained in the HiCN techniques.

Technique to prevent cuvette-related errors

1. Hold a clean cuvette only by its frosted (matt) or ridged sides. When transferring a solution to a cuvette, allow the fluid to run down the inside wall of the cuvette. This will help to avoid air bubbles in the solution. Do not fill a cuvette more than three quarters full.

2. Using a tissue or soft clean cloth, wipe clean the clear optical surfaces of the cuvette.
Carefully insert the cuvette in the colorimeter or haemoglobin meter (optical surfaces facing the light source).
Note: Ensure a solution is at room temperature before reading its absorbance otherwise condensation will form on the outside of the cuvette which will give an incorrect reading.

COLLECTION OF 24-HOUR URINE SPECIMEN

1. Sanitize your hands and assemble the equipments

2. Greet and identify the patient. Introduce yourself to patient and explain the procedure

Instruct the patient as follows:

3. When you get up the in the morning empty your bladder into the commode just as you normally do In other words, this urine is not to be saved. Make note of the time.

4. The next time you need to urinate, void the urine directly into the plastic container.

5. Tightly screw the lid onto the container, and put into refrigerator or into an ice chest.

6. Repeat steps 4 & 5 each time you urinate.

7. On the next morning, get up exactly the same time ( exactly 24hrs after beginning the test). Void into the container for the last time.

8. Put the lid on the container tightly. Return the urine collection container to the office the same morning you complete the urine collection.

Provide the patient with the collection container and written instructions.