MALT EXTRACT AGAR (MEA)

Malt Extract Agar is used for the cultivation of fungi and is not intended for use in the diagnosis of disease or other conditions in humans. It is frequently used for culturing fungi from soil, wood, basidiomycetes etc. However, clinical specimens such as skin scrapings can be processed on the medium.

It is recommended for the detection, isolation, maintenance and enumeration of yeasts and moulds such as Aspergillus brasiliensis, Candida albicans and Saccharomyces cerevisiae.

An acidic medium which will support the growth of most yeasts and molds whilst inhibiting most bacteria.

Selectivity can be increased by further lowering the pH with the addition, after sterilization, of Lactic Acid (X037).

It should be noted that excess heating of this medium together with its low pH can easily result in hydrolysis of the agar gel producing soft plates.

The medium consists of malt extract, peptone and agar.

Cultural characteristics observed after an incubation at 25-30°C for 48-72 hours at a pH of 5.4±0.2.

Further biochemical tests must be carried out for further identification

SABOURAUDS DEXTROSE AGAR (SDA)

Sabouraud’s agar is sufficient for the recovery of dermatophytes from cutaneous samples and yeasts from genital cultures.

It is nutritionally poor with acidic pH (5.6).

Peptone (Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue) provide the nitrogen and vitamin source required for organism growth in SDA. Dextrose is added as the energy and carbon source. Agar is the solidifying agent.

Not recommended as a primary isolation medium because it is insufficiently rich to recover certain fastidious pathogenic species, particularly most of the dimorphic fungi.

It does not promote conidiation of filamentous fungi.

Sabouraud’s dextrose agar (2%) is most useful as a medium for the subculture of fungi recovered on enriched medium to enhance typical sporulation and provide the more characteristic colony morphology.

Identification of fungi is performed by observing various aspects of colony morphology, characteristic microscopic structures, rate of growth, media which supports the organism’s growth, and source of specimen.

Yeasts will grow as creamy to white colonies. Molds will grow as filamentous colonies of various colors.

PREREQUISITES FOR MYCOLOGY SPECIMEN COLLECTION

  • Always consult the SOP manual of the diagnostic laboratory for the particular specimen and test.
  • Aim to collect sample in a sterile kit under aseptic conditions.
  • Ensure that you have the appropriate swab kit (alcohol swabs, gloves, transportation tube and media).
  • If it is a special or non-routine test, coordinate with the laboratory about specific requirements for collection and handling.
  • Document the following:
  1. Patient identifiers (e.g. name, age, medical record number);
  2. Sample identifiers (e.g. swab, smear, scrape, urine, blood);
  3. Location and type (e.g. lower limb wound, nail scraping, and hair sample);
  4. Date and time of collection;
  5. Deviations from standard protocol during collection (e.g. not performed under aseptic conditions if large traumatic wound);
  6. Relevant clinical information including recent and current antimicrobial therapy.
  • Ensure that adequate material (at least 2 ml of bodily fluids) is sent to the laboratory for proper yield. An inadequate specimen may lead to a false negative result.
  • Transport the sample within 2 hours and process promptly for optimum recovery of fungi.
  • If a delay is anticipated, refrigerate specimens at 4°C (exceptions: blood, bone marrow, CSF and sterile tissues should be stored at 35-37°C).
  • Ideally, collect specimens as soon as symptoms appear and whenever possible before antifungal therapy is initiated.
  • Staff must take all precautions to avoid inadvertent contamination of sample as well as for their own personal safety.

PREPARATION OF GLUCOSE STANDARD SOLUTIONS

Stock glucose standard, 100 mmol/l


1. Weigh accurately 1.8 g of dry anhydrous glucose (analytical reagent grade).
Note: To ensure the glucose is dry, heat it in an open container in an oven at 60–80 °C for about 4 hours. Remove and close the container immediately. When cool, weigh the glucose.


2. Transfer the glucose to a 100 ml volumetric flask.
Half fill the flask with 1 g/l benzoic acid and mix until the glucose is fully dissolved. Make up to the 100 ml mark with the
benzoic acid reagent and mix well.
The glucose concentration in the flask is 100 mmol/l.


3. Transfer to a storage bottle and label. When stored at 2–8 °C the stock standard is stable for about 6 months. If stored frozen in tightly stoppered containers the stock standard is stable for at least 1 year.

Working Standards

1. Take five 100 ml volumetric flasks and number them 1 to 5. Pipette accurately into each flask as follows:


Flask Stock glucose, 100 mmol/l
1 ………………………………………. 2.5 ml
2 ………………………………………. 5.0 ml
3 ………………………………………. 10.0 ml
4 ……………………………………… 20.0 ml
5 ……………………………………… 25.0 ml


2. Make the contents of each flask up to the 100 ml mark with 1 g/l benzoic acid and mix well.
The concentration of glucose in each standard is as follows:

Flask
1 = 2.5 mmol/l
2 = 5.0 mmol/l
3 = 10.0 mmol/l
4 = 20.0 mmol/l
5 = 25.0 mmol/l


3. Transfer each solution to a storage bottle and label. Store at room temperature (20–28 °C).
The working standards are stable for about 2 months

HbS SOLUBILITY TEST METHOD


Set up with the test, a negative control (HbAA) and a positive control using blood from a person with sickle cell trait (HbAS).

1. Pipette 2 ml of working reagent into a test tube approximately 13 × 77 mm.

2. Wash in (0.1 ml) of capillary blood or well mixed venous blood.
Note: When the haemoglobin is below 70 g/l (7 g/dl), use twice the volume of blood or if a venous blood sample, use plasma reduced blood (remove about half the plasma).

3. Mix well and filter through a small (5.5 cm diameter) No. 1 filter paper.

4. Note the colour of the solution (pale yellow, pink red, or dark red) and whether there is any red precipitate (insoluble reduced HbS) on the filter paper.

Findings


HbSS . . . . . . Clear pale yellow filtrate. Abundant red precipitate on filter paper

HbAS. . . . . . Clear pink filtrate. Small amount of red precipitate on filter paper
Same result will be obtained with HbSC and HbS with other Hb variant

HbAA (normal) . . . . . . . . Dark red fluid (soluble reduced Hb) with no precipitate on filter paper
Same result will be obtained with HbAC and HbAD

Reporting results

–‘Positive for sickle cell anaemia’ when result shows HbSS appearance.

– ‘Positive for sickle cell haemoglobin’ when result shows HbAS appearance.

– ‘Negative for HbS’ when result shows HbAA appearance.

See also:

HbS SOLUBILITY FILTRATION TEST


Value:

When reagents are available, this test should be performed in preference to the sickle cell slide test because it provides information about the different sickle cell disorders. In areas where both HbS
and HbD occur this test can be used to differentiate sickle cell anaemia (HbSS) from HbSD sickle cell disease.

Principle of the test

Blood is mixed in a phosphate buffer-saponin solution containing sodium dithionite and filtered. In its deoxygenated
form, HbS is insoluble. HbSS is indicated by a red precipitate on the filter paper with a pale yellow filtrate. Other forms of
haemoglobin are soluble when in a reduced state.

Reagents

● Phosphate buffer-saponin pH 7.1
Store at 2–8 C. Renew every 3 months or if it
becomes turbid.
● Sodium dithionite powder.

Making a working reagent:

– Measure 20 ml of buffer-saponin solution.
– Add 0.2 g sodium dithionite and mix gently until the chemical is dissolved.
Note: The working reagent is not stable. It can be used only on the day it is prepared.

See also:

SICKLING TEST METHOD

Negative Control: Deliver one drop of
blood from a person that does not have a sickle cell disorder on a slide marked ‘Neg Control’.
Add an equal volume of 2% sodium metabisulphite and mix. Cover with a cover glass. Exclude any air bubbles.

If a blood from a known sickle cell trait person is available, set up also a Positive Control.

3. Place the slides in a container (plastic box or petri dishes) with a damp piece of blotting paper or tissue in the bottom to prevent drying of the preparations. Close the container and leave at room temperature.

4. After 10–20 minutes, examine the patient’s preparation microscopically for sickle cells. Focus the cells first with the 10 objective and examine for sickling using the 40 objective.
Examine several parts of the preparation. Sickling often occurs quicker in one area than the other.

RETICULOCYTE COUNT TEST METHOD

1. Filter 2–3 drops of the stain into a small tube or vial.

2. Add about 4 drops of EDTA anticoagulated blood or capillary blood and mix well.
The amount of blood used is not critical. Use at least twice the volume of blood to stain if the patient is severely anaemic.

3. Incubate at room temperature for 20 minutes or 10–15 minutes at 35–37 C.

4. Mix gently to resuspend the red cells and using a capillary or plastic bulb pipette, transfer a drop of the stained blood to each of two slides. Spread to make two evenly spread thin films. Wave the slides back and forth to air-dry the films. Protect
the films from dust and insects until the count can be performed.

5. Count the reticulocytes microscopically. Use the 10 objective (with reduced condenser iris diaphragm) to check the distribution of the red cells. Select an area where the red cells can be seen individually, add a drop of immersion oil,
and examine using the oil immersion objective (open more the condenser iris diaphragm).

6. Count systematically (i.e. consecutive fields), 500 red cells (1 000 if there are very few reticulocytes), noting the number that are reticulocytes.
Calculate the percentage of reticulocytes.

Appearance of reticulocytes

Reticulocytes appear as pale green-blue stained cells containing dark blue-violet inclusions in the form of small granules, distributed irregularly. Mature red cells stain pale green-blue.

Counting reticulocytes:

A convenient method of counting reticulocytes is to reduce the size of the microscope field by inserting in each eyepiece a circular piece of black (opaque) paper which has
a punched out hole of about 5 mm.
To calculate % of reticulocytes:
– Using a hand tally counter, count a total of 500 red cells, noting on paper the number of cells that are reticulocytes (alternatively use two hand tally counters or a white cell differential counter).
– Multiply the number of reticulocytes counted by 2.
– Divide the figure by 10 to obtain the percentage figure.

See also:

Reticulocytes count principle

SOURCES OF ERROR WHEN MEASURING ESR


● Using the wrong volume of blood to anticoagulant.

● Blood not sufficiently mixed with anticoagulant.

● Clots in the blood. Even the smallest fibrin clot in the sample will invalidate the test result.

● Air bubbles at the top of the column.

● Testing blood samples at the hottest time of the day, or leaving tests in direct sunlight. Temperatures over 25°C increase sedimentation.

● Using a pipette which is not clean or not dry.

● Pipette not positioned vertically. Even slight variations from the upright increase sedimentation.

● Not checking whether the ESR stand is level on the bench.

● Placing an ESR stand on the same bench as a centrifuge where vibration will interfere with sedimentation.

● Measuring the ESR when a patient is dehydrated.

TYPES OF JAUNDICE

Haemolytic jaundice

In haemolytic (prehepatic) jaundice, more bilirubin is produced than the liver can metabolize, e.g. in severe haemolysis
(breakdown of red cells). The excess bilirubin which builds up in the plasma is mostly of the unconjugated type and is therefore not found in the urine.

Hepatocellular jaundice

In hepatocellular (hepatic) jaundice, there is a build up of bilirubin in the plasma because it is not transported, conjugated, or excreted by the liver cells because they are damaged, e.g. in viral hepatitis. The excess bilirubin is usually of both the unconjugated and conjugated types with bilirubin being found in the urine.

Obstructive jaundice

In obstructive (post-hepatic) jaundice, bilirubin builds up in the plasma because its flow is obstructed in the small bile
channels or in the main bile duct. This can be caused by gallstones or a tumour obstructing or closing the biliary tract. The
excess bilirubin is mostly of the conjugated type and is therefore found in the urine. The term cholestasis is used to describe a failure of bile flow.

See also

Bilirubin test