Flashcards in Microbiology 3: Diagnosis of Infection Deck (18):
What are the common sites where humans have normal flora?
2. Gastrointestinal tracts
3. Urogenital tract
4.Upper respiratory tract
Clinical notes guide the lab in the culturing of bacteria and fungi, how?
• Choice of media
• Temperature of incubation
• Atmospheric conditions for growth
• Length of incubation time.
What are the common ways of identification?
Gram stain – bacteria (allows determination of structure)
Wet preparation – urine (allows observation of RBCs and bacteria
Methylene blue stain – Fungi
Electron microscopy – Viruses (may allow determination of structure)
What are the common types of culture media?
• Basal media
o Liquid – e.g nutrient broth
o Solid – e.g. nutrient broth with agar(1.5%)
• Enriched media – e.g. horse blood agar (provides extra nutrition, and detection of haemolysis), chocolate blood agar (lysis of red cells needed by certain bacteria)
• Selective media (high sugar, low pH) – e.g. Saboraud’s agar, limits bacterial growth but allows yeast and fungi to grow
• Differential and selective – e.g. MacConkey’s agar, bile salts select for gut bacteria, inclusion of lactose and a pH indicator allows for differentiation between lactose fermenters and non-fermenters.
On a sample container, you should label both sides with the patient name and the patient hospital number and/or date of birth. True or false?
What are the basic principles of specimen collection?
• Ideally, specimens must be collected prior to antibiotic therapy (otherwise the sample won’t grow well). Exceptions are made for severe and life threatening conditions like meningitis and septicaemia.
• The specimen must be representative of the condition (e.g. throat swap will not isolate the organisms causing pneumonia, or MSU sample for UTI)
• Aseptic technique, sterile swabs and containers must be used to make sure the specimen is not contaminated by other microorganisms.
o Skin must be swabbed before insertion of a needle
o Once swabbed, must not touch the area, the needle, the top of the bottom into which the specimen will be inoculated
• Correct quantity
• Correct transport conditions for specimens.
• Labelling specimens correctly
• Completing the request form
What are the temperature requirements?
Generally 37 degrees (some higher like the bacteria that causes gastroenteritis)
What is the role of the microbiologist?
– processing the specimen, identification of infectious causative agent, provision of antibiotic susceptibilities (if relevant), provides reports to the clinician
At what temperature and conditions should microorganisms be stored in?
For most specimens it is essential that they are stored and transported under particular conditions.
4°C - when we want to stop the proliferation and maintain bacteria Room temperature (25°C) – allow further multiplication to see bacteria clearly
4°C – must be kept at this otherwise we will receive a false positive due to the high numbers of natural flora.
Cerebrospinal fluid (CSF):
Room temperature (25°C) – It should be completely sterile, anything that’s there must be a pathogen! So we want to multiply anything that’s there!
What are the common macroscopic observations?
Urine – cloudy, presence of blood?
-Cloudy – tends to suggest a level of bacterial cell growth
- Presence of RBCs – tends to suggest that there is damage present or the individual is on aspirin.
CSF – turbid, bloody?
- Cloudy – tends to indicate a level of bacterial cell growth, however if clear may not necessarily be a virus
- Bloody – may be a sign of bleeding or spinal cord obstruction
Sputum – saliva or sputum, mucoid, colour
- Green, yellow, blood – Damage to lung
What happens at the microbiology lab when a sample is received?
1. Specimen and patient details are registered.
2. Macroscopic and microscopic observation and a rapid test for antigens are made. This sometimes allows a preliminary report to be provided.
3. Cultures are then set up (can take 12+ house) to identify the causative agent.
4.This is followed by antibiotic sensitivity testing and the provision of a final report.
5. Serology test is done if relevant.
What might microscopic examination be useful for?
Direct microscopy may be useful for preliminary identification if the specimens are collected from a sterile site (blood, CSF, serous fluids, tissues, bladder, lower respiratory tract) or from sites where pathogens can be easily morphologically differentiated from normal flora (mouth, nose, skin, intestinal tract, genital tract etc.).
What are the rapid tests for antigens?
Rapid tests for antigen:
• Antigen detection – detection of bacterial soluble carbohydrate antigens. Agglutination with antibody (specific for the organism) coated latex particles or RBC (e.g. Strep. pneumoniae). Visible clumps of antigen means a positive result.
• Toxin detection – by the same method (toxin specific antitoxin). (e.g. clostridium difficle)
• Polymerase Chain Reaction (PCR) – a method used to rapidly copy a segment of DNA. Amplification of DNA segments specific to different pathogens allows for detection of their presence or viral load .(e.g. chlamydia trachomatis, HPV, viral load - HIV)
o DNA strands are denatured using heat
o Primers (complementary sections of DNA) anneal to the separate strands
o An enzyme called Taq polymerase facilitates synthesis of new DNA (from free nucleotides) resulting in two strands of the original DNA
o Process repeated in a thermal cycler every few minutes many times to create millions of exact copies very quickly.
What is the objective of the transport media?
Prevents specimen drying out and maintaining the viability of microorganisms.
What are the pH conditions required by different organisms?
6.5-7 pH for most bacteria, 5-6 for moulds and yeast, <4 for some bacteria
What is the role of the clinician?
– Collection of specimen, storage and transport of specimen
What follows incubation?
Following incubation, a range of different colonies may be present. Subculture of putative pathogen will give a pure culture. Once we have a pure culture we can run various tests: gram reaction, cell morphology, colony appearance, biochemical and antibody testing.