MICROBIOLOGICAL INVESTIGATIONS REQUIRED IN VARIOUS

DISEASES AND INTERPRETATION OF RESULTS

A diagnostic procedure refers to the various laboratory tests, which are prescribed
for various reasons, by the doctor or a clinician in charge of a patient. Such tests
are carried out in order to find out which particular organism is causing an obvious
infection, to confirm or eliminate a specific site or system as the focus of infection,
to monitor patient’s progress, to determine the sensitivity of the organism to
antibiotic therapy so as to administer effective treatment to the patient etc. if the
material presented to the laboratory is of poor quality, then the result of the test
reported back to the clinician will be unreliable.

A specimen is a sample, especially one used for diagnostic analysis. Collection of

specimen for laboratory investigations is one of the regular services that nurses
render to patients. It is important that the nurse should be aware of the nature and
importance of the various tests that are carried out in the laboratory.
Nurses are responsible either directly or indirectly for the collection and delivery
of most specimens obtained from those under their care. In doing so, it should
always be remembered that the aim is to assist the laboratory staff in reaching the
most appropriate conclusions from their examination of the specimen form the
patient. For this reason, scrupulous care must be taken to observe a number of
elementary rules which are designed to ensure a valid result of tests performed in
the laboratory.

Rules to observe in collecting specimen

1. A specimen should be strictly what it is, i.e. a collection of a sample of
uncontaminated material from the patient.
2. An adequate amount of only the sample should be obtained for examination.
It is better to be generous in the collection of a specimen than to subject the
patient to further discomfort.
3. Aseptic technique should be used in obtaining specimens. This involves the
preparation of the site mentioned above. But the equipment to be used must
also be sterilized. The use of antiseptic is necessary, but this should not be
allowed to contaminate the specimen.
4. Identification of the collected specimen is an essential requirement and a
vital precaution against errors, for this reason, a well-established routine
exists so that in all cases, specimens are correctly labeled. The label should
show the patient’s name, sex, age, address (ward or home), and nature of
specimen, name of consultant, provisional or final diagnosis and date.

SPECIMEN LABEL PATIENT DETAILS

Hospital No: ___________________ Contact: ________________________
Name: ___________________ Age: ___________ Sex: ________________
Ward: _______________ Specimen: ___________ Diagnosis: ___________
Date: ________________
Each specimen is accompanied by an appropriate laboratory request form which
must be completed by the clinician. A golden rule to be remembered is that
unidentified specimen is a potential source of danger as it may contain any type of
microorganism. It is important for the clinician to include information on diagnosis
and other aspect of treatment.
5. Fresh specimen should always be sent promptly to the laboratory. All
specimens should be properly sealed to avoid spillage of their contents.
6. The patient should always receive kind consideration in these procedures.
 When assisting in the collection of specimen, in which a painful procedure
must be performed, e.g. lumber puncture, it is important that the patient
should be carefully informed of the nature of the procedure and his fears
allayed before-hand.

TYPES OF SPECIMEN FOR DIAGNOSIS

1. Blood: for culture, grouping, cross matching and serological examinations.
2. Throat swab
3. Nose swaps
4. Sputum
5. Feces
6. Urine
7. Cerebrospinal fluid
8. Ear swab
9. Wound swab
10. Eye swab
11. Vaginal swab
12. Hair for fungal infection
13. Nail for fungal infection
14. Skin lesions for fungal infection
15. Semen to discover sterility.

IDENTIFICATION OF DIFFERENT ORGANISM

MORPHOLOGY AND STAINING REACTION
The forms of bacteria, their arrangement and structure are closely examined and
noted by means of microscopic view of prepared slides with specimen from
infected persons. Bacteria also have reactions to various staining methods. Gram
stain reveals the gram positive ones as well as those that are gram negative on
microscopic examination. Ziechi-Nelsons method, also demonstrates the acid-fast
staining reaction.
STAINING: It is a pre-requisite that the material to be stained must be fixed to a
slide.
REQUIREMENT FOR STAINING: Bunsen burner, wire-loop or platinum loop,
slide, lighter, specimen to be examined, fixature fluid, iodine, water, staining rake,
alcohol or 70% methylated spirit.
PROCEDURE: the platinum loop is heated until it is red hot, i.e. to sterilize it and
then allow it to cool. The first step is essential, as it will ensure that no pathogen is
introduced into the material to be examined. This is followed by introducing the
loop into the material to be examined and carefully spreading the contents of the
wire-loop onto the middle of the slide.
The loop is withdrawn and immediately heated until red hot again so that it will be
sterilized. The film of the slide is carefully dried by gently holding it high over a
Bunsen flame. Finally, the film is fixed by softly passing the slide through the
Bunsen flame three times, care is being taken to avoid breaking the slide. The slide
is then cooled and the material is ready for staining with the appropriate stain.

THE GRAM-STAINING (STEPS)

1. The film of the material to be examined is prepared as described above;
sterility of the platinum loop should be maintained.
2. Cover the film with crystal violet solution and allow acting for about 30
seconds.
3. Cover the film with lugol iodine to form a complex or mordant with crystal
 violet, allow to stay for 30secs.
4. Wash off with H2O.
5. Pour a neutral decolorizer e.g. alcohol or 70% methylated spirit. Allow to
stay until transelvent (about 3secs.)
6. Wash off with H2O.
7. Pour counter stain solution of safranin or neutral red and leave for about 1
minute.
8. Wash off with H2O.
9. Allow the slide to dry in air before examine under the microscope on
application of immersion oil with x 100 objective.

INTERPRETATION OF WHAT IS OBSERVED AND CLINICAL

IMPORTANCE
A. Staining: Gram-positive organism retains the crystal violet dye color and is
seen as purple structures under the microscope. E.g. Staphylococcus aureus,
Streptococcus subtilis e.t.c.
Gram-negative organisms are decolorized but are counter stained with red
color in order to render them visible under the microscope, e.g. Ecoli,
Salmonella, Gallinanum e.t.c.
B. Cultural characters: includes the growth requirements and the appearance
of the cultures to the naked eye (e.g. observation of the microorganism on
the surface of solid culture medium, such as nutrient or growing cultures,
already introduced into fluid and solid media in an incubator, temperature at
0
37 c.
C. Biochemical reactions: some biochemical tests have been found useful to
differentiate various bacteria. Majority of such tests are designed to detect
the presence of enzymes in the organism which bring about a specific
chemical reaction. An example of such tests involve the observation of
whether or not a growth of bacterium in liquid nutrient medium will ferment
particularly sugars (like glucose and mannitol) using them as a source of
energy. E.g. of the organism seen by this test are the flagella group of
organism.
D. Antigenic characters: this involves the identification of species of bacteria
by specific antibody reaction observed in serological tests. The use of anti-
sera, (prepared from immunized laboratory animals) which can agglutinate
the homologous organism in laboratory studies. The effect can be seen by
the naked eye.
E. Pathogenicity and Toxigenicity: this refers to an artificial way of
introducing bacteria in cultures into experimental animals by inoculation to
detect the actual bacteria present in the culture e.g. are tubercle bacilli
inoculated and the intradermal injection of culture material into guinea pigs.
F. Antibiotic Sensitivity: this is a process of testing an organism for its ability
to grow on artificial nutrient media.
G. Cultivation of bacteria: cultivation is a controlled growth of identification.
This is supported by the principle that if microorganisms are given the
conditions for growth, they can be identified.

One of the most important methods of identifying microorganism is by

observing their growth on artificial food materials, which are prepared in the
laboratory. This is to identify a particular medium that a certain organism can
grow and multiply. The food material is called culture medium and the growth
itself is known as culture.
CULTURATION OF BACTERIA: a culture medium must satisfy the
following criteria:
 1. It must contain appropriate nutrients in form of protein, carbohydrate,
minerals and some percent of H2O.
2. It must be at a correct PH for the growth of the particular organism, i.e.
either neutral or slightly alkaline in reaction, except acidophilus organisms.
3. The medium must be sterilized before introduction of organisms.
CULTURE MEDIA: a culture media is a kind of food material produced in
order to mimic the growth of microorganisms in vitro, before a substance can be
considered to be a culture media, it must possess the following conditions such
as all essential nutrition, ions and moisture, maintain the correct PH and
osmotic pressure and neutralize any toxic material produced. There are three (3)
kinds of culture media: liquid, semi-solid and solid.
1. Liquid (Broth media): this is a liquid media that does not gel after cooling.
2. Semi solid media: this is a media which are formed by adding a small
amount of agar.
3. Solid (Agar media): this is a media that solidifies after cooling. There are
many media that can be classified under this: basic media enrich media,
selective media, indicator media, transport media and identification media.
a. Basic media: these are simple media such as nutrient agar and nutrient
broth that will support the growth of microorganism that do not have
special nutritional requirement.
b. Enriched media: are required for the growth of organisms with exacting
growth requirement such as PH influence e.g. Neisseria species.
c. Enrichment media: this is usually applied to fluid selective media which
inhibit the growth of one organism to allow the growth of another to be
clearly demonstrated.
d. Selective media: this is a media used in selecting the growth of organism
by altering or changing the PH of the media.
e. Indicator media: these are media to which dye or other substances are
added to differentiate microorganisms. This is done by incorporation of
an indicator which change color when acid is produced following
fermentation of a specific carbohydrate e.g. Macon key.
f. Transport media: these are mostly semi-solid media that contain
ingredients to prevent the over growth of commensals and ensure the
survival of aerobic and anaerobic pathogens when specimen cannot be
cultured immediately.
g. Identification media: these include media to which substrate or chemicals
are added to help identify bacteria isolated on primary culture.
There are numerous common nutrient found in diagnostic laboratory for
culturing purpose. They are as follows: nutrient agar, blood agar, Macon key
agar, Salmonella and Shigella agar, Thiosulphate citrate bile salt (TCBS),
cook meat media, Baird Parker agar, mannitol salt agar, Lawenstain Jensen
media, and Selenite F media etc.

Some common problems concerning the laboratory diagnosis establishment are

discussed as under:

PYREXIA OF UNKNOWN ORIGIN (PUO)

Peterson and Beeson defined PUO as illness of 3 weeks’ duration temperature
exceeding 38.3°C on several occasions. Invariably diagnosis is not established
even after one week’s stay in the hospital. The causes of PUO are:
Acute (short duration)
1. Enteric fever
2. Amebic hepatitis
3. Urinary tract infections
4. Malaria
5. Sub-acute bacterial endocarditis
6. Brucellosis
7. Typhus fever
Chronic (long duration)
1. Brucellosis
2. Pulmonary tuberculosis
3. Typhus
4. Sub-acute bacterial endocarditis
5. Kala-azar.
Investigations are done as under:
1. Urine for microscopic examination especially for pus cells. In case of
presence of pus cells culture and drug sensitivity is done.
2. Blood examination:
a. TLC and DLC shows leukocytosis in urinary tract infection, sub-acute
Bacterial endocarditis and amebic hepatitis. Leukopenia is a feature of enteric fever
and malaria. Lymphocytosis may be observed in brucellosis.
b. RBC count and hemoglobin estimation indicates anemia.
c. Peripheral blood film may show malarial parasite.
d. Bone marrow may show LD bodies.
3. Sputum examination is done for the demonstration of acid-fast bacilli by
direct, concentrated and culture methods.
4. Stool examination for trophozoites and cysts of Entamoeba histolytica is
done. One may also look for Charcot-Leyden crystals.
5. Blood culture is done by collecting 5 to 10 ml of blood from the patient
aseptically using autoclaved syringe. Blood culture bottles are used
containing 50 ml of broth. Glucose bile broth is used for enteric fever,
glucose broth for sub-acute bacterial endocarditis or other pyogenic
organisms and liver infusion broth (Castaneda’s media) for brucellosis.
These bottles are incubated at 37°C and sub cultured on solid media (blood
agar and MacConkey agar plates) after 24 hours, 48 hours, 7 days and 14
days. For enteric look for non-lactose fermenter colonies (NLF) which are
Gram negative bacilli, oxidase negative, ferment glucose and mannitol,
Indole and VP negative, and MR plus citrate positive. Final confirmation for
enteric bacilli is done by slide agglutination with specific antisera. For sub-
acute bacterial endocarditis subculture is done on blood agar plate after 2, 7
and 30 days and studies the colonies of Streptococcus viridans,
Staphylococcus albus, and Streptococcus fecalis. For brucellosis incubation
is done in 5 to 10 percent CO2 and look for Gram negative coccobacilli,
non-lactose fermenter, non-motile and oxidase negative bacilli. Confirmation
is done by agglutination with specific antisera.
6. Serology: Widal’s test is done for enteric fever. Agglutination test for
brucellosis may be done. Weil-Felix test using OX2, OX19 and OXk antigen
is done for typhus fever. Formal gel test can be done for kala-azar.
7. Pus may be examined microscopically for trophozoites of Entamoeba
histolytica and necrotic debris.

SORE THROAT
ETIOLOGY
a. Membranous
1. Corynebacterium diphtheriae
2. Candida
3. Vincent’s angina
b. Non-membranous
1. Streptococcus pyogenes
2. Staphylococcus aureus
3. Pneumococcus
4. Pertussis
5. Corynebacterium diphtheriae
6. Adenovirus
7. Rhinovirus

Collection of Specimen
Throat swab is taken aseptically using tongue depressor. It is necessary that
collection of throat swab should be undertaken under proper light. It is advisable to
take two throat swabs. If membrane is present one must make it a point to remove
part of the membrane because chances of recovery of causative organism are more
from membrane.

Culture
Immediately after the collection of specimen, inoculate it on blood agar, blood
tellurite and Loffler’s serum slope. Incubate these plates at 37°C for 12 to 48 hours.
In the meantime smears from throat swabs are prepared, and then Gram’s and
Albert staining is done. In stained smear, look for Gram-positive bacilli, which are
thin, slender, pleomorphic with metachromatic granules showing Chinese latter
arrangement. These characters are suggestive of Corynebacterium diphtheriae,
Gram-positive cocci in chains (streptococcus), in clumps (staphylococcus) and if in
pairs (pneumococcus). Budding yeast cells are found in candida. In Vincent’s
angina we find curved and spiraled organisms, i.e. fusiform bacilli. In diphtheria
cases we may find small, convex colonies on blood agar plates. On Loffler serum
slope there is abundant growth which is moist, cream colored or pigmented. Blood
tellurite agar is selective and indicator medium (black colonies) showing
differentiation among gravis, intermedius and mitis. Sugar sets are used. Sugar
media is prepared in Hiss serum. In case of gravis, starch and glycogen are
fermented without formation of gas. Pathogenicity tests are useful like Elek’s test,
animals’ inoculation test, e.g. rabbit and guinea pig in which material may be
injected intradermally or subcutaneously. In Staphylococcus aureus cases we find
colonies 2 to 3 mm in diameter, smooth, glistening butyrous, and opaque and
golden yellow showing beta hemolysis on blood agar plate. They are catalase
positive and Gram-positive cocci arranged in clusters. They exhibit pathogenicity
tests like coagulase production, mannitol fermentation, lipase, DNase and
phosphatase production. The Streptococcus pyogenes is Gram-positive cocci
arranged in chains. They are catalase negative. On blood agar plate they are small,
low convex, semitransparent, discrete colonies showing beta hemolysis. They are
matt to mucoid when freshly isolated. Biochemically they ferment lactose, glucose,
sucrose and mannitol with only acid production. Most common Lancefield group
responsible for throat infection is A. Pneumococcus shows alpha hemolysis. The
colonies are small, flat and transparent. They show positive bile solubility test,
ferment insulin and optichin sensitivity test is positive.
They may kill mice when injected intraperitoneally.

MENINGITIS
Causes
A. Bacterial:
1. Meningococci
2. Pneumococci
3. Hemophilus influenzae-b
4. Streptococci
5. Staphylococci
6. Coliform organism
7. Mycobacterium tuberculosis
8. Treponema pallidum
9. Leptospira
b. Viral:
1. Mumps
2. Coxsackie
3. Echo
4. Herpes simplex
5. Lymphocytic choriomeningitis
6. Poliomyelitis
7. Arbovirus
c. Fungal:
1. Cryptococcus
2. Coccidioides
3. Histoplasma capsulatum

Collection of specimen
Specimen required for investigation of meningitis is cerebrospinal fluid. It is
collected aseptically in an autoclaved container by performing lumbar puncture
and specimen should be arranged to be transported to laboratory quickly. If delay is
inevitable then specimen may be kept at 37°C for not more than 4 hours. It should
never be kept in refrigeration or icebox.

Processing and culture of specimen

In the laboratory gross examination of cerebrospinal fluid is done to note color,
turbidity and any deposit or clot. Microscopic examination is also done for white
cell count. Some fluid is immediately transferred into glucose broth. Fluid left
behind is centrifuged. Supernatant may be used for biochemical test like protein,
sugar and chloride. Smear is prepared from deposit and stained by Gram’s Method.
Deposit is also used for culture on blood agar plate. Similarly, subculture from
glucose broth may be done on blood agar plate. After 37°C incubation for 48 hours
plates are studied and colonies are identified by various biochemical reactions.
Drug sensitivity is also determined. In tuberculosis cases cerebrospinal fluid
frequently contains clot (cobweb or spider web). Smear and then Ziehl Neelsen
stain from this clot may show acid-fast bacilli. Lowenstein Jensen medium may be
used for the culture of Mycobacterium tuberculosis and for animal inoculation.
Cerebrospinal fluid deposit may be injected into guinea pig. For demonstration of
fungus microscopic examination is done. Subsequently for cultural purposes
Sabouraud’s agar media may be used. Some serological tests may be helpful in
diagnosing fungal etiology. For the demonstration of virus as etiological agent
electron microscope, tissue culture and serological techniques may be undertaken.