33.3 Bacterial Diagnosis Flashcards
What are Koch’s postulates, as a concept?
Four criteria that were designed to establish a causative relationship between a microbe and a disease.
What are the four Koch’s postulates?
That the specific micro-organisms are…
1. isolated from all animals suffering the disease
2. grown in pure culture from animal
3. injected into healthy animals, and disease is reproduced in the second animal
4. recovered in pure culture
What is MacConkey agar and what is it useful for?
- A medium used to differentiate between Gram-negative bacteria based on their ability to digest lactose.
- It contains a pH indicator and lactose.
- If the bacteria can digest carbohydrates, acid is produced and the result is pink colonies.
- If the bacteria cannot digest carbohydrates, acid is not produced and the result is colourless colonies.
What does a pink MacConkey agar indicate?
The bacteria can digest carbohydrates, so acid is produced and the result is pink colonies.
What does a colourless MacConkey agar indicate?
The bacteria cannot digest carbohydrates, acid is not produced and the result is colourless colonies.
What is often added to MacConkey agar and why?
- Bile salts
- This is because MacConkey is useful for distinguishing between Gram-negative bacteria that can grow in the intestine, so bile salts are used to kill any Gram-positive bacteria and those not adapted to living in the intestines
Describe the colour and specific selectivity of XLD agar
Pinkish-red, selective for Shigella (appears pink/ red) and Salmonella (appears black)
Why does Salmonella appear black on XLD agar?
Able to convert the sodium thiosulfate in the agar to H2S
- which then reduces the ferric ammonium citrate in the agar to iron sulfide (black)
Why does Shigella appear pink on XLD agar?
Cannot ferment one or more of the agar sugars
Why is it necessary to use selective media for identifying pathogens from clinical specimens?
Need to inhibit normal microorganisms from microbiota
Where may clinical specimens of bacteria be taken from?
- Nasopharynx
- Blood
- CSF
- Tissue
Which protective measures may be put in place for handling pathogens safely?
- Wear PPE to cover skin
- Wear goggles
- May have vaccination if highly dangerous
Why can specimens from sites that are usually sterile be inoculated without selective agents?
There should be no microbiota that would otherwise also be picked up
What are the purposes of inoculating bacteria on an agar plate?
- Enables any contamination to be easily detected
- Allows well-separated, single colonies to be isolated and grow - forming ‘pure’ cultures
What are the two main types of media in which bacteria are grown?
Solids and liquids
What does preliminary identification of bacteria include?
Gram staining, identification of colony and cellular morphology.
Gram negative bacteria will stain…
Pink
What are the key properties of the cell wall of Gram negative bacteria?
- Two membranes
- Periplasm in between
- Lipopolysaccharide
Gram positive bacteria will stain…
Dark purple/blue
What are the key properties of the cell wall of Gram positive bacteria?
- Single membrane
- Relatively exposed peptidoglycan wall
What are the methods to test antibiotic susceptibility/resistance of bacteria?
- Disc diffusion assay
- E-strip
- Restriction enzyme mapping analysis
How does a disc diffusion assay measure antibiotic susceptibility?
- Paper discs with known antibiotic concs are placed on a plate of bacteria
- No growth = sensitive to antibiotic
- And area of no growth reflects degree of sensitivity
How does an E-strip measure miminum inhibitory concentration (MIC)?
- Inert strip impregnated with a gradient of antibiotic is placed on the surface of a bacterial lawn
- Intersection between strip and start of growth shows MIC
How does MacConkey agar select for Gram-negative, gut-adapted bacteria?
Contains crystal violet and bile salts that…
- inhibit growth of most Gram+ bacteria (except straph and enterococci)
- select for gut bacteria, since bile salts destroy Gram- bacteria that are not gut-adapted
What are the different tests for identifying bacterial species that you need to know?
- Gram staining
- Cellular morphology and colony morphology
- Catalase
- Coagulase
- Oxidase
- Streptococcal carbohydrate antigen grouping (Lancefield typing)
What is bile salt testing useful for?
Media containing bile acids are helpful for identifying bacteria that survive in the small intestine, a feature of many Enterobacteriaceae.
What is the catalase test and what is it used for?
- A test for the presence of a bacterial enzyme that breaks down hydrogen peroxide.
- A drop of hydrogen peroxide is applied to a bacterial colony using a glass capillary tube. A positive result is the appearance of bubbles on the surface of the colony.
- The ability of bacteria to degrade hydrogen peroxide can directly impact where they can live and the types of infection they cause.
What is the haemolysis test and what are the different results?
It is where a blood agar is used to test the haemolytic properties of an unknown bacterium.
What is a presumptive diagnosis associated with?
Empirical therapy –> treatment based on experience + begun on the basis of a clinical “educated guess” in the absence of complete or perfect information.
What is Lancefield typing and what is it used for?
- It is a.k.a. Streptococcal carbohydrate antigen grouping
- It is used to differentiate between the different Streptococcus species, which each show different carbohydrate antigens.
- It is an agglutination assay. The latex particles coated with specific antibodies will only agglutinate in the presence of the homologous antigen, and will remain in suspension otherwise.
- The results as stated as Group A, Group B, etc.
What is the oxidase test and what is it used for?
A test that detects cytochrome oxidase, a transmembrane protein complex of the respiratory electron transport chain, which is present in aerobic organisms that can use oxygen as a terminal electron acceptor.
What is the urease test and what is it used for?
- It is a test for the urease enzyme
- It is used to test for bacteria such as H. pylori
- Urease can split urea to generate ammonia and CO2, leading to a rise in pH. The test includes a pH indicator, typically phenol red, which turns red when the pH rises above 8.
What is the coagulase test and what is it used for?
- It is a test for the coagulase enzyme
- It is used to differentiate between Staphylococcus aureus and a coagulase-negative Staphylococcus
- A positive Coagulase test is detected by aggregation/clumping of the latex particles.
Some bacterial species apart from Salmonella produce H2S. Why do these not produce black colonies on XLD agar?
They do not produce lysine decarboxylase, which the Salmonella use to produce an adequately alkaline environment for H2S production.
Draw the flowchart of diagnostic tests for identifying unknown bacteria.
Summarise the different lab tests required to test for various Streptococcus species.
- Gram-positive
- Coccus
- Negative catalase
- Alpha haemolysis + Optochin sensitive -> Streptococcus pneumoniae
- Beta haemolysis + Lancefield type A -> Streptococcus pyogenes
Summarise the different lab tests required to test for Staphylococcus aureus.
- Gram-positive
- Coccus
- Positive catalase
- Positive coagulase
Summarise the different lab tests required to test for Clostridia species.
- Gram-positive
- Bacillus
Summarise the different lab tests required to test for Escherichia coli.
- Gram-negative
- Bacillus
- Growth in bile salts
- Positive lactose fermentation (MacConkey)
- Positive indole test
Summarise the different lab tests required to test for Shigella species.
- Gram-negative
- Bacillus
- Growth in bile salts
- No lactose fermentation (MacConkey)
- Negative oxidase test
- Negative urease test
- No H2S production
Summarise the different lab tests required to test for Salmonella species.
- Gram-negative
- Bacillus
- Growth in bile salts
- No lactose fermentation (MacConkey)
- Negative oxidase test
- Negative urease test
- Positive H2S production
What is the role of the diagnostic laboratory?
To make and confirm diagnoses, monitor treatment, antibiotic susceptibility testing, bacterial typing, provision of information for local, national and international epidemiology.
What is a presumptive diagnosis based on?
It is based on clinical signs, patient history, and local epidemiology.
How is a presumptive diagnosis refined?
Diagnosis + treatment are refined following lab tests.
Summarise the different lab tests required to test for Neisseria species.
- Gram-negative
- Coccus
- Positive oxidase test
