33.3 Bacterial Diagnosis

Question 1

What are Koch’s postulates, as a concept?

Answer 1

Four criteria that were designed to establish a causative relationship between a microbe and a disease.

Question 2

What are the four Koch’s postulates?

Answer 2

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

Question 3

What is MacConkey agar and what is it useful for?

Answer 3

• 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.

Question 4

What does a pink MacConkey agar indicate?

Answer 4

The bacteria can digest carbohydrates, so acid is produced and the result is pink colonies.

Question 5

What does a colourless MacConkey agar indicate?

Answer 5

The bacteria cannot digest carbohydrates, acid is not produced and the result is colourless colonies.

Question 6

What is often added to MacConkey agar and why?

Answer 6

• 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

Question 7

Describe the colour and specific selectivity of XLD agar

Answer 7

Pinkish-red, selective for Shigella (appears pink/ red) and Salmonella (appears black)

Question 8

Why does Salmonella appear black on XLD agar?

Answer 8

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)

Question 9

Why does Shigella appear pink on XLD agar?

Answer 9

Cannot ferment one or more of the agar sugars

Question 10

Why is it necessary to use selective media for identifying pathogens from clinical specimens?

Answer 10

Need to inhibit normal microorganisms from microbiota

Question 11

Where may clinical specimens of bacteria be taken from?

Answer 11

• Nasopharynx
• Blood
• CSF
• Tissue

Question 12

Which protective measures may be put in place for handling pathogens safely?

Answer 12

• Wear PPE to cover skin
• Wear goggles
• May have vaccination if highly dangerous

Question 13

Why can specimens from sites that are usually sterile be inoculated without selective agents?

Answer 13

There should be no microbiota that would otherwise also be picked up

Question 14

What are the purposes of inoculating bacteria on an agar plate?

Answer 14

• Enables any contamination to be easily detected
• Allows well-separated, single colonies to be isolated and grow - forming ‘pure’ cultures

Question 15

What are the two main types of media in which bacteria are grown?

Answer 15

Solids and liquids

Question 16

What does preliminary identification of bacteria include?

Answer 16

Gram staining, identification of colony and cellular morphology.

Question 17

Gram negative bacteria will stain…

Answer 17

Pink

Question 18

What are the key properties of the cell wall of Gram negative bacteria?

Answer 18

• Two membranes
• Periplasm in between
• Lipopolysaccharide

Question 19

Gram positive bacteria will stain…

Answer 19

Dark purple/blue

Question 20

What are the key properties of the cell wall of Gram positive bacteria?

Answer 20

• Single membrane
• Relatively exposed peptidoglycan wall

Question 21

What are the methods to test antibiotic susceptibility/resistance of bacteria?

Answer 21

• Disc diffusion assay
• E-strip
• Restriction enzyme mapping analysis

Question 22

How does a disc diffusion assay measure antibiotic susceptibility?

Answer 22

• 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

Question 23

How does an E-strip measure miminum inhibitory concentration (MIC)?

Answer 23

• 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

Question 24

How does MacConkey agar select for Gram-negative, gut-adapted bacteria?

Answer 24

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

Question 25

What are the different tests for identifying bacterial species that you need to know?

Answer 25

* Gram staining * Cellular morphology and colony morphology * Catalase * Coagulase * Oxidase * Streptococcal carbohydrate antigen grouping (Lancefield typing)

Question 26

What is bile salt testing useful for?

Answer 26

Media containing bile acids are helpful for identifying bacteria that survive in the small intestine, a feature of many Enterobacteriaceae.

Question 27

What is the catalase test and what is it used for?

Answer 27

* 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.

Question 28

What is the haemolysis test and what are the different results?

Answer 28

It is where a blood agar is used to test the haemolytic properties of an unknown bacterium.

Question 29

What is a presumptive diagnosis associated with?

Answer 29

Empirical therapy --> treatment based on experience + begun on the basis of a clinical "educated guess" in the absence of complete or perfect information.

Question 30

What is Lancefield typing and what is it used for?

Answer 30

* 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.

Question 31

What is the oxidase test and what is it used for?

Answer 31

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.

Question 32

What is the urease test and what is it used for?

Answer 32

* 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.

Question 33

What is the coagulase test and what is it used for?

Answer 33

* 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.

Question 34

Some bacterial species apart from Salmonella produce H₂S. Why do these not produce black colonies on XLD agar?

Answer 34

They do not produce lysine decarboxylase, which the Salmonella use to produce an adequately alkaline environment for H₂S production.

Question 35

Draw the flowchart of diagnostic tests for identifying unknown bacteria.

Answer 35

Question 36

Summarise the different lab tests required to test for various Streptococcus species.

Answer 36

* Gram-positive * Coccus * Negative catalase * Alpha haemolysis + Optochin sensitive -\> Streptococcus pneumoniae * Beta haemolysis + Lancefield type A -\> Streptococcus pyogenes

Question 37

Summarise the different lab tests required to test for Staphylococcus aureus.

Answer 37

* Gram-positive * Coccus * Positive catalase * Positive coagulase

Question 38

Summarise the different lab tests required to test for Clostridia species.

Answer 38

* Gram-positive * Bacillus

Question 39

Summarise the different lab tests required to test for Escherichia coli.

Answer 39

* Gram-negative * Bacillus * Growth in bile salts * Positive lactose fermentation (MacConkey) * Positive indole test

Question 40

Summarise the different lab tests required to test for Shigella species.

Answer 40

* Gram-negative * Bacillus * Growth in bile salts * No lactose fermentation (MacConkey) * Negative oxidase test * Negative urease test * No H₂S production

Question 41

Summarise the different lab tests required to test for Salmonella species.

Answer 41

* Gram-negative * Bacillus * Growth in bile salts * No lactose fermentation (MacConkey) * Negative oxidase test * Negative urease test * Positive H₂S production

Question 42

What is the role of the diagnostic laboratory?

Answer 42

To make and confirm diagnoses, monitor treatment, antibiotic susceptibility testing, bacterial typing, provision of information for local, national and international epidemiology.

Question 43

What is a presumptive diagnosis based on?

Answer 43

It is based on clinical signs, patient history, and local epidemiology.

Question 44

How is a presumptive diagnosis refined?

Answer 44

Diagnosis + treatment are refined following lab tests.

Question 45

Summarise the different lab tests required to test for Neisseria species.

Answer 45

* Gram-negative * Coccus * Positive oxidase test