21BTT359 Biomedical Diagnostics

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Microbial
Diagnostics
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Principles of Microbial Diagnosis
▪ Specimen Selection, Collection, and Processing

➢ selected on the basis of signs and symptoms,

➢ should be representative of the disease process

➢ should be collected before administration of antimicrobial

agents.

▪ The specimen amount and the rapidity of transport to the

laboratory influence the test results.
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Microbiologic Examination
▪ Direct Examination and Techniques: for gross pathology; e.g. Microscopy,
Immunofluorescence, immuno-peroxidase staining, etc.

▪ Culture: Isolation of infectious agents with specialized or pathogen selective media.

▪ Microbial Identification: Colony and cellular morphology may permit preliminary

identification. Growth characteristics under various conditions, utilization of
carbohydrates and other substrates, enzymatic activity, immunoassays, and genetic
probes are also used.

▪ Serodiagnosis: A high or rising titer of specific IgG antibodies or the presence of

specific IgM antibodies may suggest or confirm a diagnosis.

▪ Antimicrobial Susceptibility: Microorganisms, particularly bacteria, are tested in vitro

to determine whether they are susceptible to antimicrobial agents.
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Specimen Selection, Collection and
Processing
▪ Specimens selected for microbiologic examination
should reflect the disease process and be collected in
sufficient quantity to allow complete microbiologic
examination.

▪ The number of microorganisms per milliliter of a body

fluid or per gram of tissue is highly variable, ranging
from less than 1 to 108 or 1010 colony-forming units
(CFU)
 Microbiologic Examination
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Test Specimen Pathogen
Gram Stain Blood, CBSF, skin (wound), Bacteria – Gram positive (purple), Gram
respiratory secretions, urine, negative (pink)
urethral or cervical exudates
Giemsa Blood Malaria, spirochetes and other blood parasites
staining Chlamydia, Yersinia pestis, Histoplasma spp.
Acid fast Respiratory secretions Bacteria - Mycobacterium tuberculosis
staining
India ink wet CBSF Encapsulated microorganisms, especially
mount Cryptococcus neoformans
KOH wet Respiratory secretions, skin Fungi and fungal structures
mount
Eosin wet Faeces Intestinal parasites
mount
Methylene Faeces, blood, Respiratory Leukocytes, viable cells
blue stain secretions and other body fluids Histopathology of cancer tissues
Gram Staining and Acid Fast Staining
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Selective Media
▪ Used for the growth of only
selected microorganisms.
▪ For example, if a microorganism is
resistant to a certain antibiotic,
such as ampicillin or tetracycline,
then that antibiotic can be added to
the medium in order to prevent
other cells, which do not possess
the resistance, from growing.
Differential media or
indicator media
▪ Distinguish one microorganism
type from another growing on the
same media.
▪ This type of media uses the
biochemical characteristics of a
microorganism growing in the
presence of specific nutrients or
indicators (such as neutral red,
phenol red, eosin y, or methylene
blue) added to the medium to
visibly indicate the defining
characteristics of a microorganism.
 z Selective Media
▪ Eosin methylene blue (EMB) that contains methylene blue – toxic to
Gram-positive bacteria, allowing only the growth of Gram negative
bacteria.

▪ YM (Yeast and Mold) Agar and Broth which has a low pH, deterring
bacterial growth.

▪ MacConkey agar for Gram-negative bacteria.

▪ Hektoen enteric agar (HE) which is selective for Gram-negative

bacteria.
 z Selective Media
▪ Mannitol salt agar (MSA) which is selective for Gram-positive
bacteria and differential for mannitol. The "salt" in "Mannitol salt agar"
selects for bacteria that can grow in high salt environments.
Staphylococci thrive on the medium, largely because of their
adaptation to salty habitats such as human skin.
▪ Terrific Broth (TB) is used with glycerol in cultivating recombinant
strains of Escherichia coli.
▪ Xylose lysine desoxyscholate (XLD) selective for Gram-negative
bacteria
▪ Buffered charcoal yeast extract agar, which is selective for certain
gram-negative bacteria, especially Legionella pneumophila.
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Differential Media
▪ Blood agar (used in strep tests), which contains bovine heart
blood that becomes transparent in the presence of haemolytic
bacteria - useful in classifying streptococcal species.

▪ Eosin methylene blue (EMB), which is differential for lactose and

sucrose fermentation.

▪ MacConkey (MCK), which is differential for lactose fermentation

▪ Mannitol salt agar (MSA), which is differential for mannitol

fermentation.

▪ X-gal plates, which are differential for lac operon mutants.

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Growth of Viruses and Chlamydiae

▪ Chlamydiae (obligate intracellular bacteria) and viruses are

cultured in cell culture systems, but virus isolation
occasionally requires inoculation into animals, such as
suckling mice, rabbits, guinea pigs, hamsters, or primates.

▪ Rickettsiae may be isolated with some difficulty and at some

hazard to laboratory workers in animals or embryonated
eggs.
CULTURE OF BACTERIA, FUNGI & VIRUS
Bacteria
Spherical or irregular colonies
pH 5-9 (optimum 7),
Acidophiles (<5.55, Sulfolobus
spp.), Alkaliphiles (pH 8.0 -
10.5, Vibrio cholerae)
Nutrient agar, tryptic soy agar,
and brain heart infusion agar,
Nutrient broths or lysogeny
broth (LB) medium
Mesophiles (20 °C - 45 °C)
e.g., E. coli, Salmonella spp.,
and Lactobacillus spp.
Fungi
Filamentous or rhizoid colonies
pH 5-6
Sabouraud dextrose agar
(SDA), Potato dextrose Agar,
Malt extract, Hichrome Agar,
Dichloran Glycerol Agar
Mesophiles (5–35°C), optimum
25 - 30°C
Virus
Morphology as per host cells,
visible evidence of cell
damage, as newly formed
virions spread
As per conditions of host cells
As per the media required for
culturing the host cells, or
cultured in embryonated hen's
eggs, or laboratory animals
As per conditions of host cells
CULTURE OF BACTERIA, FUNGI & VIRUS
BACTERIAL GROWTH CURVE AND ITS SIGNIFICANCE
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Thank you!!

Revise:

✓ General procedure for

microbial diagnostics

✓ Staining of microbes

✓ Culture of bacteria, fungi and

virus