D.Y.

PATIL UNIVERSITY, NAVI MUMBAI

SCHOOL OF BIOTECHNOLOGY AND BIOINFORMATICS
PRACTICAL MANUAL PREPARATION
Programme- MTI (M.tech Integrated)

Semester-6

Practical Title- Isolation and identification of microorganisms causing respiratory

tract infection

Aim- To isolate and study the causative agent/ pathogen causing

infection of the respiratory tract.

Principle- The respiratory tract is broadly divided into the upper i.e the orthopharynx and
the nasopharynx and the lower ie larynx, trachea, bronchi and the alveolar sacs of the lungs.
The middle ear may be considered as part of the upper respiratory tract as it is connected to
the posterior pharynx via the Eustachian tube.
Samples for the respiratory tract infections include swabs, sputum or aspirates.
Interpretations must take into account the presence of normal flora of the concerned part.
Samples for lower respiratory tract infection should be processed as soon as possible and if
there is going to be a delay, it should be refrigerated at 2 - 8C. special care should be
taken for samples got by invasive techniques as a repeat sample may be difficult to get.
Specimen type and collection:
1. Swabs: A swab is collected by scrubbing it on the affected area and immediately
placing it in a sterile tube. In case of a throat swab, care should be taken not to
touch the lateral walls of the buccal cavity or the tongue to minimize contamination
by commensals. Any purulent discharge should also be sampled.
2. Sputum : Early morning sputum samples should be obtained because they contain
pooled overnight secretions in which pathogenic bacteria are more likely to be
concentrated.
3. Transtracheal aspirate: This is generally collected in from debilitated patients who
cannot expectorate a sputum. It is also used if there is a clinical indication of
bacterial pneumonia but routine sputum samples have not shown any causative
organism.
4. Bronchial aspirate is recommended if a patient is suspected to have deep seated
pulmonary lesions.
Specimen transport :
Respiratory samples should normally be processed immediately as the pathogens are
sensitive to temperature and there is a likelyhood of overgrowth of the recommensals if
there is a delay. Samples should be placed in transport medium (eg Stuarts medium) and
refrigerated if the laboratory cannot process in 2 hrs.
Parential etiologies suspected:
Streptococcus spps, Haemophilus influenza. Klebsiella spps, Bacteroides spps,
Corynebacterium diphtheriae, Niesserria spps, Bordetella pertussis, Mycobacterium
tuberculosis etc. Staphylococcus aureus can be present in the nasal swab of carriers.

RequirmentsDay 1
1. Sample: throat aspirate sample/ sputum sample
2. Gram staining:
1

a. Crystal violet.
b. Distilled water/ tap water.
c. Grams iodine.
d. alcohol
e. Saffranin
f. Oil, grease-free glass slide, compound microscope, burner.
3. Isolation of pathogen:
a. Nichrome loop.
b. Sterile nutrient agar plates
c. Sterile differential media plates
d. Sterile selective media plates
e. Incubation 37C 24 hrs
Day 2
1. Sterile Nutrient broth
2. Gram staining requirements
3. Biochemical kit
4. Special test requirements
Day 3
Reagents for biochemicals

Procedure:
Day 1:
1. Perform the gram staining of the given sample to find the gram nature of the pathogen.
2. Depending on the gram nature, select three media for the isolation of the pathogen. The media should
a) Nutrient agar for isolating the pathogen and show the presence of pigment producer
b) Differential medium
c) Selective medium
3. Incubate the plates at 37C 24 hrs.
Day 2:
1. Select a well isolated colony from each of the three plates, and study the colony characteristic of thes
organisms.
2. Perform the gram staining of the isolates.
3. Carry out the special test if required.
4. Inoculate the colony selected from Nutrient agar plate in fresh sterile nutrient broth and incubate at 3
half an hour.
5. Inoculate 50 l of this culture in each well of the biochemical test kit.
6. Incubate at 37C for 24 hours.
Day 3:
Add specific reagents to the biochemical test kit as per the manual and compare the observations with th
provided in the manual.
For example : If the causative agent is S. aureus/S. pyogenes
Media : St Nutrient agar (all pathogens can grow and pigment producers can be distinguished)
St Blood agar ( observing the type of hemolysis)
St Salt mannitol agar (selective medium)

Result-

Conclusion / Discussion