LAB 5

Question 1

what is tryptic soy agar considered as

Answer 1

non-selective media

Question 2

colony morphology of S.aureus and S.epidermidis on TSA

Answer 2

• circular
• 1mm
• convex
• entire
• cream/white
• wet
• smooth
• dull
  –> good growth

Question 3

mannitol salt agar

Answer 3

• both a selective and a differential culture medium
• formulated for the growth of gram-positive bacteria belonging to the staphylococcal group

Question 4

appearance of S.aureus and S.epidermidis on mannitol salt agar

Answer 4

growth = good
colony morphology = circular, 1mm, cream, smooth, raised
agar colour:
s. aureus = yellow
s. epidermidis = pink

Question 5

what are the selective components in mannitol salt agar

Answer 5

7.5% concentration of sodium chloride

Question 6

what group of bacteria is mannitol salt agar selective for

Answer 6

staphylococcus

Question 7

list the differential components of mannitol salt agar

Answer 7

mannitol salt
phenol red agar

Question 8

how does mannitol salt agar differentiate between the group of bacteria

Answer 8

• If an organism can ferment mannitol, an acidic by-product is formed that will cause the phenol red in the agar to turn yellow
• Most pathogenic staphylococci will ferment mannitol
• Most non-pathogenic staphylococci will not ferment mannitol
  eg:
  stappylococcus aureus turns yellow = can ferment mannitol

Question 9

what is blood agar

Answer 9

• an enriched and differential media used to provide increased nutrients for microbial growth and detect haemolytic activtiy

Question 10

B haemolysis

Answer 10

the complete lysis of red blood cells in the agar, resulting in a completely clear zone around the bacterial colonies

Question 11

A haemolysis

Answer 11

the partial lysis of red blood cells, resulting in a greenish zone in the agar

Question 12

Y lysis

Answer 12

non haemolytic = doesn’t produce zones of lysis

Question 13

results of blood agar

Answer 13

S. aureus
- clear zone = complete lysis
= B haemolysis
S. epidermidis
- no lysis
= Y haemolysis

Question 14

what are the differential components in blood agar

Answer 14

sheeps blood 5%

Question 15

staphylococci characteristics

Answer 15

• gram positive
• facultative aerobic cocci
• characteristically form aggregates of cells, resembling bunches of grapes

Question 16

procedure of the catalase test

Answer 16

1. Hold one end of a capillary tube between your thumb and middle finger.
   
   2. Dip the other end of the tube into 3% hydrogen peroxide, the liquid will rise part way up the tube by capillary action.
   3. Stopper the end of the tube you are holding with your index finger, this will prevent the liquid dripping out.
   4. Touch the end holding the liquid to an isolated colony on an agar plate so as to transfer a small amount of colony to the end of the tube.
      Look for vigorous bubbling in the capillary tube.

Question 17

result of the catalase test for S.aureus and S. epidermidis

Answer 17

both positive
- produce the enzyme catalase
S. aureus = releases O2

Question 18

results of gram stain for S.aureus and S. epidermidis

Answer 18

• gram positive
• regular
• cocci
• clusters
• 1um
• regular form
  –> not able to tell them apart from gram reaction

Question 19

what does the coagulase test, test for

Answer 19

–> used to differentiate S.aureus from other commonly isolated staphylococci
Coagulase exists in two forms: ‘Bound’ to the cell wall or free which is liberated by the cell wall
Bound coagulase = detected by slide test
free coagulase = detected by tube test

Question 20

coagulase test procedure

Answer 20

1. Place 20 µL of distilled water or saline on a clean microscope slide.
2. Make a thick suspension of the organism to be tested in the drop of saline. Do not let it dry.
3. Drop 20 µL of plasma onto the suspension, and then gently rock the slide.

Question 21

coagulase test results

Answer 21

s. aureus = postitive, has clumping
s. epidermidis = negative

Question 22

what three tests or observations can be used to differentiate S.aureus from s.epidermidis

Answer 22

• coagulase
• plating on mannitol salt agar
• haemolytic on blood agarw

Question 23

what are two virulence factors that S.aureus exhibits

Answer 23

• coagulation
• B-haemolysis

Question 24

what are enrichment techniques

Answer 24

techniques used to isolate the various kinds of bacteria that co-exist in a particular habitat

Question 25

procedure of culturing from the nasal swab

Answer 25

1. Label the mannitol salt agar plate and the nutrient broth + 7.5% NaCl with the appropriate information. 2. Collect a nasal sample by moistening a swab with distilled water (remove excess water by pressing the swab against the side of the tube). Insert the swab inside one nostril and press it firmly against the mucosa for at least one minute. 3. Use the streak plate procedure to inoculate this sample onto the mannitol salt agar plate. Use the swab to inoculate the initial inoculum then use a sterile loop to complete the streaking. 4. Invert the plate and place in the container at the end of your bench for incubation at 35±2°C for 18-24 hours. 5. Collect a sample from your other nostril, using a fresh swab. 6. Immerse this swab into the nutrient broth + 7.5% NaCl, break the stick, leaving the swab in the medium. 7. Place in the rack at the end of your bench. Incubate at 35±2°C for 18-24 hours.

Question 26

what is the benefit of using an enrichment broth such as nutrient broth + 7.5% NaCL

Answer 26

- used for the cultivation of many species of non-fastidious microorganisms - especially those that are presented in low numbers

Question 27

what are replicate organism detection and counting (RODAC) plates used for

Answer 27

the detection and enumeration of microorganisms present on a wide variety of surfaces including the skin - the design of the dish allows pouring of a raised convex surface of the culture medium for total surface contact of the area being sampled

Question 28

RODAC procedure

Answer 28

1. Label the RODAC plates with the appropriate information and the sample site. 2. Press one plate against your forehead for 10 seconds. 3. Press the other plate against your forearm for 10 seconds. 4. Invert the plates and place in the container at the end of your bench. Incubate at 35±2°C for 18-24 hours.

Question 29

procedure for hand hygiene

Answer 29

1. Label the TSA plates with the appropriate information. 2. Moisten a swab with distilled water and collect a sample from one hand by firmly rolling the swab over the palm. Take about 15 seconds to do this to make sure all bacteria in the area are transferred to the swab. 3. Gently rub the swab across the entire surface of one TSA plate to transfer as much of the sample as possible to the plate. 4. Wash your hands - one out of each pair is to wash with normal soap, the other is to wash with antibacterial soap or use the alcohol sanitiser. Cleaning agent used: normal soap 5. Collect another sample from the same hand, and inoculate the other TSA plate. 6. Invert the plates and place in the container at the end of your bench. Incubate at 35±2°C for 18-24 hours.

Question 30

bacteriophage typing

Answer 30

--> a phenotypic method of typing S. aureus and is based on different bacterial strains displaying different susceptibility to lysis by a panel of bacteriophages (a method of identifying and classifying bacteria based on their susceptibility to specific bacteriophages)

Question 31

how can bacteria acwuire antibiotic resistance

Answer 31

through mutation or exchange of genetic material among same or closely related species

Question 32

disc diffusion (qualitative methods)

Answer 32

categorise a bacterial isolate as sensitive, intermediate or resistant to a particular antibiotic

Question 33

the kirby bauer test

Answer 33

= a disc diffusion procedure used to determine the sensitivity or resistance of pathogenic aerobic and facultative anaerobic bacteria to various antimicrobial compounds - tests the antimicrobial susceptibility of bacteria based on the size of zones of inhibition on a bacterial lawn culture around discs impregnated with an antimicrobial agent - When an organism is susceptible to the antibiotic in a disc, a zone of inhibition around the disc is present. Beyond the zone is an unaffected area of normal growth

Question 34

what is the diameter of the inhibition zone in the kirby bauer test an indication of

Answer 34

the amount of drug in the disc and susceptibility of the microorganism to that drug

Question 35

results of mueller-hinton (kirby bauer) test

Answer 35

amoxicillin = susceptible ciprofloxacin = susceptible penicillin = resistant --> recommend the the drug with the largest diameter

Question 36

MIC

Answer 36

= minimum inhibitory concentration - the minimum concentration of antibiotic that inhibits bacterial growth ie the lowest conc. in the series that is clear

Question 37

MLC

Answer 37

= minimum lethal concentration - the lowest conc. of antibiotic that has killed the test organism; this is shown by the absence of growth