UNIT SEVEN

Microbiological method

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 Specimen collection
- If pathogens are to be isolated successfully the
following points must be considered: type of specimen,
time of collection, collection techniques
1. Type of specimen
The correct type of specimen to collect will depend on
the pathogens to be isolated
Eg - a cervical not a vignal swab - N.gonorrhoeae
-Sputum, not saliva - respiratory pathogen

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2. Time of collection:
- Consider the right time at which the organism
proliferate maximum and before the pt take
antimicrobial drug
- Example: Urine & sputum is best collected soon
after a patient wakes, blood for culture is best
collected when a patients temp. begin to rise.

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 Specimen collection cont..

3. Collection techniques:
- the laboratory should issue written instructions to all
those responsible for collecting specimens including
the staff of wards, out patient clinics, and health
centers

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 Specimen collection cont..
The following precautions apply to the collection of
most microbiological specimen
- A strictly sterile (aseptic) procedure - normally
sterile (Ex. blood, CSF)
- Avoid contaminating discharges or ulcer material
with skin commensal
- The swab used to collect the specimen must be
sterile and free from any antibacterial substances

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 Specimen collection cont..
Collect specimens in sterile, leak- proof, dry
containers, free from all traces of disinfectant
Containers
- must be clean, but need not be sterile for sputum and
stools
- must be easy for patients to use
Patients must be instructed how to collect aseptically

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Macroscopic feature of the sample must be reported

Color of the sample
Presence of pus, blood, mucus
The appearance of urine, pus, vaginal discharge,
faeces, CSF

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Each specimens must be clearly labeled with

The date and time of collection
The patients- name, age, Hospital number, Ward or
Health center
Slides with one end frosted should be used to label
with a lead pencil

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Specimens containing dangerous pathogens

Those delivering, receiving, and examining
specimens must be informed if a specimen is likely to
contain highly infectious organisms.
Such a specimen should be labeled HIGH RISK, and
when ever possible carry a warning symbol such as a
red dot or star, which helps for easy recognition

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 Specimen collection cont..

Specimens which should be labeled as HIGH RISK

include:
Sputum likely to contain M. tuberculosis
Fecal specimen that may contain V.cholerae or S.
typhi
Fluid from ulcers or pustules that may contain
anthrax bacilli or treponemal.
Specimens from patients with suspected HIV
infection, hepatitis, viral hemorrhagic fever, or
plague.

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 Specimen collection cont..

Preservatives and transport media for

microbiological specimen
If there is any delay in specimen delivery use chemical
preservative.
This will help to prevent organisms from dying due to
- Enzyme action
- Change of PH
- Lack of essential nutrients

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 Staining
A. Grams stain
- Gram stain divides bacteria in to two physiologic
groups, gram positive &gram negative
- Gram positive bacteria retain the primary stain ,
appear violet or deep purple in color
- Gram negative bacteria A gram negative bacteria
have a high lipid content which dissolves in the
decolorization process and stain with counter stain
(Pinkish red)

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 Staining

B. Acid fast staining

Zeehel- Nelson technique is used to stain
Mycobacterium species including M. tuberculosis
and M. leprae.
The Mycobacterial cell is difficult to stain by gram
staining because they posses a waxy envelopes and a
special method has to be used.
Besides being difficult to stain, once it is stained the
organism is hard to decolorize.

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 Culture & Biochemical and
susceptibility test
- Culture is artificial cultivation of microorganisms in the
laboratory
- Culture is important for isolation and identification of
microorganisms based on different characteristics such as
Colony characteristics
Ability to produce pigments
Hemolysis
To metabolize different nutrients
Gas production
Drug susceptibility test to select appropriate medication

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