Module 1 Practical

Question 1

When an organism has been isolated and reported, what part of a microbiology specimen report (lab results) do doctors place the most significance to?

Answer 1

Identification

Question 2

In what situations would an accurate species identification be important? List two reasons.

Answer 2

• overt pathogens always require accurate speciation
• ID is important for ongoing and recurrent infections
• epidemiological accuracy - references would never get updated

Question 3

What is the relationship between specimen quality and the time interval between collection and processing?

Answer 3

• Specimen quality decreases with time
• The longer it takes for a specimen to reach the laboratory, the more difficult it may be to isolate or identify a pathogen
• Overgrowth with normal flora
• Non-survival of anaerobes or fastidious (fussy) organisms

Question 4

What is the ideal maximum elapsed time between collection and processing?

Answer 4

• Ideally, specimens should be received within 2 hours
• Large Hospitals (should be very rapid since labs. are usually on site)
• Private and small hospitals (from several hours to overnight/24-36 hours)

Question 5

What are two advantages of placing a swab in transport media?

Answer 5

• the semisolid agar provides moisture to prevent drying out
• maintains pH
• Use of transport media for swabs
• preserves the viability of bacteria without multiplication

Question 6

List two specimens that have a high priority once in the lab?

Answer 6

• High priority to CSF, tissue, blood cultures, sterile fluids

Question 7

What is the significance of seeing PMN cells in a wet preparation or stained smear?

Answer 7

• The presence of polymorphonuclear WBCs is a good indicator of infection.

Question 8

What does the presence of large numbers of epithelial cells in either a sputum or urine sample indicate?

Answer 8

Epithelial cells in sputum & urine = indicate likely contamination with Normal Flora

Question 9

Why are CHOC plates always incubated in CO2?

Answer 9

• ALWAYS incubated in CO2 to accommodate Haemophilus and Neisseria

Question 10

List 4 guidelines for ensuring a good quality microbiology specimen.

Answer 10

• The aim is to transport the specimen while maintaining the sample in as close to the original state as possible
• Ideally, specimens should be received within 2 hours
• Large Hospitals (should be very rapid since labs. are usually on site)
• Private and small hospitals (from several hours to overnight/24-36 hours)
e.g. MSU collected on a weekend (stored in a fridge until collected Mon AM)
e.g. Blood collected in the country (i.e. Bunbury) for a referred test in Perth
• Paperwork and specimen should be shipped together (in a bio. bag)

• Avoid extremes of temperature
• Leads to overgrowth or death of the micro-organisms
• Can destroy the integrity of sample e.g.. faecal fat in summer (see next)
• Urine, faeces, sputum, swabs, catheters can be stored at 4˚C (fridge)
But, faeces at 4 ˚C is no good for detecting amoebic parasites
• CSF to be kept at 37˚C (to detect Nagleria fowleri)
• Specimens suspected of containing anaerobes should be kept at RT

Question 11

List 4 reasons why a laboratory might reject a microbiology specimen?

Answer 11

Each laboratory will have its own set of guidelines
- Reject a collected specimen from being processed if the specimen has been collected into formalin
- Unlabeled specimens
- Samples with illegible labeling
- Mismatched details
• name on the specimen is different to that on the paperwork
- Insufficient material and/or leaking specimens
- Specimens for anaerobes that have been stored at 4˚C
- Inappropriate specimen collected
• e.g. dry swab (Chlamydia PCR type) with a request for MC+S (see next)
- Non-sterile container for a urine or aspirate
- Salivary sputum samples
- First void urine samples for UTI investigations
- Any specimen collected into formalin (blue top used for histology specimens)
- Specimens that have experienced an excessive delay in getting to the laboratory
• e.g. specimen left in the fridge over the weekend

Question 12

Other than MHA, for each type of agar that was used in this practical exercise, describe whether it is selective only, differential only, selective and differential or enriched.

Answer 12

Blood agar: differential - used to distinguish pathogenix bacteria based on effect of bacterial enzymes (haemolysins)
MacConkey agar: selective and differential - isolate and differentiate enteris based on ability to ferment lactose
Mannitol salt age: selective and differential - high salt concentration,, selects for members of staphylococcus, tolerate high saline levels

Question 13

Describe how MacConkey agar No 3. works? Your answer should detail the important components and their function as well as explain which organisms grow/do not grow.

Answer 13

MAC: selective and differential medium designed to selectively isolate gram negative and enteric bacilli and differentiate them based on lactose fermentation. Contains bile salts, lactose and crystal violet dyes - known to inhibit growth of gram positive bacteria. Staphylococcus = gram positive thus unable to grow

Question 14

Describe how MSA agar works?

Answer 14

MSA: encourages growth of certain bacteria groups while inhibiting others. If an organism can ferment mannitol, acidic byproduct is formed causing phenol red in agar to turn yellow.
Bile salts and crystal violet dye makes it selective (due to salt concentration) - bile salts inhibit growth in gastro-intestinal tract
Lactose makes it differential - carbohydrate, neutral red indicator (always include in explanation)

Question 15

Why should the Gram result for Candida albicans in Table 1 be recorded as “large purple staining ovoid cells”?

Answer 15

Yeast is not a bacteria and doesn’t contain a gram +/- cell wall

Question 16

Why do Gram negative bacteria stain pink?

Answer 16

Don’t retain crystal violet-iodine complex, hence, when they become decolourised they are easily stained by carbol fuchsin

Question 17

Why don’t Gram positive bacteria decolourise during the alcohol step of the Gram staining procedure?

Answer 17

Gram positive retain crystal violet-iodine complex

Question 18

Which discipline of taxonomy is responsible for giving an organism a scientific name?

Answer 18

Nomenclature - labelling/naming of the groups and the members

Question 19

What is an obligate aerobe?

Answer 19

Obligate aerobe - grows in air, requires molecular oxygen for growth, will not grow anaerobically

Question 20

Give an example of an obligate aerobe (genus and species)?

Answer 20

Mycobacterium tuberculosis

Question 21

What is an obligate anaerobe?

Answer 21

Obligate anaerobe - will only grow in the complete absence of oxygen

Question 22

Give an example of an obligate anaerobe (genus and species)?

Answer 22

Clostridium perfringens

Question 23

What is a facultative organism?

Answer 23

Facultative anaerobe - will grow in the presence or absence of oxygen

Question 24

Give an example of a facultative organism (genus and species)?

Answer 24

Escherichia coli

Question 25

What does microaerophilic mean?

Answer 25

Microaerophilic - requires oxygen for growth but at a lower concentration than is present in the atmosphere (6-16% O2, 2-10% CO2) - generated using a gas pack

Question 26

Name a genus of bacteria that has this characteristic (Q8)?

Answer 26

Haemophilus influenzae - haemophilus

Question 27

What is a capnophile?

Answer 27

• Capnophilic (carboxyphilic) - grow best with increased carbon dioxide (2.5%-10% CO2 & about 15%O2)

Question 28

Name a genus of bacteria that has this characteristic (Q10)?

Answer 28

Campylobacter coli - campylobacter

Question 29

List the genus and species of an organism that is acid fast.

Answer 29

• Mycobacterium and Nocardia are positive

* Have acid fast cell wall

Question 30

Which genera of bacteria produce spores?

Answer 30

Bacillus and clostridium are positive

Question 31

Why do you have to be careful when performing a catalase test on a colony taken from BA?

Answer 31

Getting blood media onto the slide can cause false positives

Question 32

What precautions do you need to take when performing an oxidase test?

Answer 32

• Don’t use iron containing loops or wires (false positive results)
• Use platinum wires or toothpick

Question 33

The Gram Stain

Answer 33

The gram stain is the most widely used staining procedure in bacteriology. It is called a differential stain since it differentiates between gram-positive and gram-negative bacteria. Bacteria, which stain purple with the gram staining procedure, are termed gram-positive; those, which stain pink, are said to be gram-negative.
Gram-positive and gram-negative bacteria stain differently because of fundamental differences in the structure of their cell walls. The bacterial cell wall serves to give the organism its size and shape as well as to prevent osmotic lysis. The material in the bacterial cell wall, which confers rigidity, is peptidoglycan. The gram-positive cell wall appears thick and consists of several interconnecting layers of peptidoglycan as well as some teichoic acids. Generally, 80% -90% of the gram-positive cell wall is peptidoglycan. The gram-negative cell wall, on the other hand, contains a much thinner, single layer of peptidoglycan and is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, and lipoprotein. Only 10% - 20% of the gram-negative cell wall is peptidoglycan.
Ø Gram positive bacteria
- thick layer of peptidoglycan with teichoic acid cross linkages
- resist decolourisation (crystal violet-iodine complex intact)
- blue/purple colour
- coccus/bacillus
Ø Gram negative bacteria
- thin layer of peptidoglycan
- decolourisation due to disruption of lipid rich outer membrane and thin layer of peptidoglycan
- colourless until counterstained
- pink colour
- coccus/bacillus/curved/helical

Question 34

Specialised media

Answer 34

Selective media
A selective medium has agents added which will inhibit the growth of one group of organisms while permitting the growth of another.
Differential media
A differential medium contains additives that cause an observable colour change in the medium when a particular chemical reaction occurs. They are useful in differentiating bacteria according to some biochemical characteristic. In other words, they indicate whether or not a certain organism can carry out a specific biochemical reaction during its normal metabolism.
Enrichment media
An enrichment medium contains additives that enhance the growth of certain organisms. This is useful when the organism you wish to culture is present in relatively small numbers compared to the other organisms growing in the mixture.
Combination selective and differential media
A combination selective and differential medium permits the growth of one group of organisms while inhibiting the growth of another. In addition, it differentiates those organisms that grow based on whether they can carry out particular chemical reactions.

Question 35

Explain the principle of action of the catalase test?

Answer 35

(how does it work, what is happening?)
• Catalase is an enzyme which catalyses the breakdown of hydrogen peroxide to produce oxygen and water

• Mix colony material with hydrogen peroxide and observe for the production of effervescence
• Weak positive reaction If inoculum is contaminated with RBC (BA)
• staphylococci (GPC) = positive
• streptococci and enterococci (GPC) = negative
• Use any Staphylococcus sp. as a positive control
• Hydrogen peroxide at RT deteriorates - always check