2.
General Principles of Laboratory Diagnosis of Bacterial
diseases.
Exercise 2.1:
1) Suggest suitable clinical samples to be collected in the following infections/diseases.
Type of infection/disease Suitable Clinical sample to be collected
Conjunctivitis, keratitis, endopthalmitis Conjunctival swab, corneal scrapings, Aqueous or Vitreous
fluid.
Otitis media Swabs from outer ear , Aspirate from inner ear
Oral and dental infections Oropharyngeal swab,
Meningitis Cerebrospinal fluid. (CSF)
Skin and soft tissue infections Aerobic:- Pus or exudate, wound swabs, aspirates from
abscess and tissue bits
Anaerobic :- Aspirates, tissue specimens (Swabs not
satisfactory)
Upper respiratory tract infections Throat swab with membrane over the tonsil,
(Pharyngitis, tonsillitis, diphtheria) nasopharyngeal swab, per-nasal swab
Lower respiratory tract infections Sputum, endotracheal aspirate, Broncho alveolar lavage
(Bronchitis, Pneumonia, pulmonary (BAL), Protected specimen brush (PSB) and lung biopsy.
tuberculosis)
Septicemia, Paired blood culture specimens: - Collected aseptically by
Endocarditis two-step disinfection of skin, first with alcohol followed by
chlorhexidine.
8-10 ml of blood (for adult) collected in blood culture bottle
Diarrhea, Dysentery, Food poisoning Stool (Mucus flakes), rectal swab.
Genital infections (Gonorrhea, syphilis, Urethral swab, cervical swab – for Urethritis, Exudates from
chancroid etc.) genital ulcers.
Urinary tract infections Midstream urine, suprapubic aspirated urine, Catheterized
patient – collected from the catheter tube, after clamping
distally and disinfecting not from urobag.
Diseases requiring Blood (2ml/Investigation), collected by minimal asepsis
serological/immunological tests (one-step skin disinfection with alcohol) Collected in
vacutainer.
2. Describe the precautions to be taken while collecting samples for anaerobic
culture.
Following samples should be avoided for anaerobic cultures:-
Bronchial washings,
Sputum, stool, throat swab, urine, rectal swab etc.,
Clinical specimens suitable for anaerobic culture:-
Bile, biopsy of endometrial tissue, collection of purulent specimens from deep wound, using sterile
syringe/needle.
The above samples should be immediately sent to laboratory, if delay is expected, transport in
Anaerobic Transport media (ATM).
3. Enumerate the culture media and methods used for anaerobic culture.
Anaerobic culture media Anaerobic culture methods
1. Robertson’s cooked meat media (RCM) 1. Evacuation & Replacement
2. Thiogylcollate broth. a. Manual method ( McIntosh & Fildes
3. Anaerobic blood agar. jar).
4. Egg yolk agar. b. Automated method.
5. Neomycin blood agar. 2. Absorption of oxygen by chemical
methods.- GasPak system
3. Reducing agents.
4. What are the criteria for sample rejection?
1. Improper labelling of the specimen.
2. Incomplete specimen related or clinical information.
3. Suboptimal sample – sample leaking from container
Insufficient quantity
Inappropriate sample for test required
5. What are the general principles to be followed for specimen collection?
1. Standard precautions to be followed- while collecting & handling samples
2. Appropriate site of sample collection
3. Appropriate time of sample collection
4. Sufficient quantity of specimens.
5. Appropriate collection devices.
6. Samples to be collected before the antibiotics are administered.
7. Container should be properly labelled.
6. Label the blank parts of the packed clinical specimen in the following picture
7. Write the general guidelines for specimen transportation.
Proper packing
Transport time should not exceed two hours. (CSF, Suprapubic aspirates, body fluids should
reach the laboratory within 15 minutes)
If there is any delay- Special preservative /holding media
Urine – boric acid (24 hours)
Use of transport medium wherever required.
Stool- Cary Blair medium
8. What are the important and essential components of specimen label?
Patient name,
Age,
Gender,
OPD/IPD No,
Department,
Ward/unit,
Date & time of collection,
Sample
9. Following is the sample of specimen request form. Please fill the details as per the
clinical case discussion in this practical exercise.
10. Enumerate the steps used in bacterial culture.
Collection of the samples following standard guidelines,
Inoculation of the samples on to the appropriate culture media.
Incubation of the inoculated culture media at appropriate temperature and pH
Identification of the organisms by appropriate methods,
Antibiotic sensitivity testing
11.Write the methods used for bacterial identification
Conventional methods:-
Culture smear:- Gram’s stain,
Motility testing:- by Hanging drop method
Biochemical identifications:- coagulase, catalase, Indole, citrate, Urease TSI
Automated system:-
MALDI-TOF (VITEK-MS)
VITEK -2
12.Write in brief on professional conduct pertaining to the clinical samples
Consent for sample collection and type of investigation should be explained to the patient.
Privacy and confidentiality,
Sample should be collected by following all the standard guidelines
The collected clinical sample should be sent to laboratory under proper transportation condition in time
along with completely filled with requisition form.
The sample should be processed in time and the report should be made available in time.
The sample should be discarded following biomedical waste management (BMW) regulations
AETCOM Exercise
1. Biopsy sample of a critical patient getting rejected because it was collected in a formalin saline
Ans:-
Improper collection of sample - sample should have been collected in Saline because formalin
kills the organisms and the clinical sample will be unsuitable for culture.
Delay in diagnosis/misdiagnosis – Can be detrimental to patient life
Financial and psychological burden for the patient
2. Left over serum sample getting used for research without informed consent
Ans:- It is unethical to use any patient sample for research without their informed consent
3. Sample is accompanied with request form with incomplete information/ wrong patient credentials
Ans:-
Sample should be accompanied with requisition form which is duly filled with all relevant information
Incomplete requisition form will lead to misdiagnosis/wrong diagnosis.
e.g., 1. Urine: - Sample should be processed within 30 minutes. Hence time of sample collection should be
mentioned.
2. Widal test:- which detects significant titer of antibodies in the 2nd week of the illness (fever). Hence
it is important to mention duration of fever.
4. Undue delay in testing and reporting of a sample requiring emergency testing.
Ans:-
Undue delay in testing of clinical sample requiring emergency testing will be detrimental to life of
patient.
e.g., CSF: - Gram stain - Pneumococci/ Cryptococcus
Negative stain - Pneumococci/ Cryptococcus
Cryptococcal antigen detection
All these tests can be done 30 minutes of reported in time which can be life saving to the patient.
