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2.2 Microbiology > Day 1: Question 2 - Inoculating Tests > Flashcards

Day 1: Question 2 - Inoculating Tests Flashcards

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1
Q

How do you carry out a DN’ase test?
(8)

A

Ask for DN’ase agar containing 0.2% DNA

Split plate down the middle

Split one half into halves again for the positive and negative control

Spot inoculate test strain and controls

Incubate at 37 degrees Celsius

After incubation put int fume cupboard and flood plate with 1M HCL

Allow HCL to permeate for 10 minutes then carefully pour off excess

Observe plate for distinct clear zones around colonies (S. aureus) or cloudy DNA precipitates (non S. Aureus)

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2
Q

What are your controls for the staph DN’ase test?

A

Positive clear distinct zone of clearance = S. aureus

Negative cloudy = S. epidermidis or S. saprophyticus

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3
Q

What are your controls for mannitol salt agar?

A

positive yellow colonies = S. aureus

negative pink/red colonies = S. epidermidis or S. saprophyticus

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4
Q

What are your controls for SAIDE agar?

A

Positive pink colonies = S. aureus

Negative non-coloured colonies = S. epidermidis or S. saprophyticus

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5
Q

How do you carry out a Staphaurex Plus test
(6)

A

Ask for latex reagent, reaction card, control latex (some sticks to mix)

Shake the latex reagent to mix

Dispense one drop of test latex onto one of the circles and one drop of control reagent onto another circle

Using a loop (wooden end of swab/stick) pick up and smear 2-3mm of Staph growth onto a circle and mix this in the control latex reagent

Using a clean loop/stick proceed in the same way with the Test Latex

Pick up and rock the card for 20 seconds

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6
Q

What are your controls for the Staphaurex test

A

Positive agglutinating control = S. aureus

Negative = coag neg staph = S. epidermidis or S. saprophyticus

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7
Q

How do you test for novobiocin resistance?

A

Ask for blood agar and novobiocin disc and (bijou bottle? containing nutrient broth - don’t necessarily need this you can just streak out a plate and add disk)

Lawn inoculum

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8
Q

What are your controls for the novobiocin resistance test?

A

Positive resistant = S. saprophyticus

Negative susceptibility = S. Epidermidis/ S. aureus

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9
Q

How do you carry out an optochin susceptibility test
(7)

A

Ask for blood agar, optochin disk x3, bijuo jar of sterile water and a swab x3

Divide a blood agar into three section, half for your test and two quarters for positive and negative control

Prepare a suspension of test organism and controls using a reduced volume of sterile water

Lawn inoculate your test and controls

Place one optochin disc in centre of test inoculum and the controls

Incubate overnight at 37 degrees

Examine for a zone of inhibitions

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10
Q

What are your controls for optochin susceptibility?

A

Positive susceptibility = Streptococcus pneumoniae
Negative resistance = Streptococcus viridans

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11
Q

How do you prepare your bacteria for the Lancefield agglutination test?
(4)

A

Dispense 0.4ml of Oxoid Streptococcus Extraction Enzyme into a labelled test tube

Select 2-5 test colonies and emulsify in the enzyme preparation

Incubate for 10 minutes at 37 degrees Celsius in a water bath -> make sure to shake vigorously at 5 mins in

Remove and allow to cool to room temp

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12
Q

How do you carry out the Lancefield grouping test
(6)

A

Ask for a reaction card, sticks, latex reagent A, B, C, D, F and G,

Ask for S. pyogenes or Enterococcus control?

Ask for (pre-mixed?) enzyme (and test) extract??? -> don’t know if this will be done for you previously or you will have to do it yourself

Add 1 drop of latex reagent A, B, C, D, F, G to a circle on your reaction card

Look for agglutination

Carry out this test using the positive control first so you know the reagents are working

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13
Q

How do you carry out a Bacitracin susceptibility test?
(6)

A

Ask for blood agar, x3 bijou jars full of sterile water, and x3 bacitracin discs

Divide a blood agar plate into three sections, one for test, two for controls

Prepare a suspension of test and controls using a reduced volume of sterile water

Lawn inoculum of test and controls

Place one bacitracin disc in the centre of each inoculum

Incubate overnight at 37 degrees

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14
Q

What are your controls for the bacitracin test?

A

Positive susceptibility = S. pyogenes
Negative resistance = any other staph or Enterococcus e.g. E. faecium

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15
Q

What are your controls for MacConkey agar?

A

Positive pink colonies = Enterococcus
Negative yellow colonies = gamma haemolytic strep e.g. Strep bovis

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16
Q

How should you carry out a bile susceptibility test?

A

Ask for a bile aesculin agar plate, E.faecalis + control, Strep pyogenes negative control

Spot inoculate

Incubate at 45 degrees !!!!

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17
Q

What are your controls for bile aesculin agar test?

A

Positive blackening of medium = enterococcus e.g. E. faecium
Negative no colour change = S. pyogenes

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18
Q

What four sugars are used in the TAXO sugar utilisation test?

A

Glucose

Maltose

Sucrose

Lactose

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19
Q

How do you carry out a TAXO sugar utilisation test
(6)

A

Ask for a x2 chocolate agar plate, x2 TAXO sugar discs and x2 bijou jar containing sterile water

Prepare a heavy suspension of the test isolate using 5ml sterile water

Lawn the inoculum over the entire surface of a chocolate agar plate

Using sterile forceps place the TAXO discs on the inoculum

Use N. lactamica as your control

Incubate at 37 degrees -> add phenol red after incubation

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20
Q

What should you use as your control for TAXO sugar test?

A

N. lactamica

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21
Q

How do you carry out the catarrhalis disk test
(3)

A

Ask for two catarrhalis disc, ask for a M. catarrhalis control

using a sterile wooden applicator to rub several colonies of test or control onto catarrhalis disc

Wait for 2 minutes to observe a colour change

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22
Q

What are your controls for catarrhalis disk test?

A

Positive = blue-green colour development = M. catarrhalis
Negative = no colour development within 2 mins = Neisseria

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23
Q

What feature of catarrhalis can be seen from a colony?

A

Hockey-puck colonies

24
Q

What are you controls for Moraxella DN’ase test?

A

Positive = zone of clearance = M. Catarrhalis
Negative = cloudy = Neisseria

25
Q

How do you carry out a PEMBA test?

A

Ask for PEMBA, a B. cereus control and a B. subtilis control

Streak inoculate

Incubate at 37 degrees Celsius

26
Q

What are your controls for PEMBA?

A

Positive lecithinase = clearance

Negative lecithinase = no clearance

Positive mannitol = yellow colonies

Negative mannitol = peacock blue colonies

B. cereus = blue colonies with clearance
B. subtilis = yellow colonies with no clearance

27
Q

How do you carry out the motility test
(6)

A

Apply a small amount of vaseline to the four corners of a clean coverslip

Add one drop of broth culture of test organism is added to centre of the slip

The cavity slide is inverted and centred over the drop of culture and lowered onto the coverslip

The slide is quickly turned right side up so that the hanging drop is suspended in the well

Place the slide on the microscope under low intensity

Locate the edge of the drop

28
Q

What are your controls for motility test?

A

Positive control = motile = any bacillus other than anthacis
Negative control = non motile = B. anthracis is the only non motile Bacillus

29
Q

How do you carry out the Trehalose and Urease biochemical tests
(4)

A

Ask for a trehalose peptone water sugar bijou and a urease agar bijou

Label a blood agar plate to be used as a purity plate

Inoculate the trehalose peptone water sugar and sequentially inoculate the urease agar and then the purity plate without flaming

Incubate each at 37 degrees Celsius

30
Q

What is the best way of inoculating the trehalose agar?
(2)

A

Tilt the liquid media to the right and gently rub the inoculated loop on the left side of the tube just below the meniscus

Turn the tube upright and gently shake to inoculate the broth

31
Q

What is the best way of inoculating the urea slope?

A

Zig-zag across the surface of the agar and stab the slope with the loop

32
Q

What are your controls for the trehalose and urease tests?

A

Positive trehalose = yellow = C. ulcerans
Negative trehalose = pink = C. diptheria

Positive urease = pink = C. ulcerans
Negative urease = yellow = C. diptheria

33
Q

How do you carry out a Tinsdale test

A

Ask for a Tinsdale agar, C. diphtheriae and a diphtheroid such as haemophilus, neisseria, staphylococcus and strep

Incubate at 37

34
Q

What are your controls for the Tinsdale test?

A

positive = black colonies with brown halo = C. diptheriae or C. ulcerans
negative = black colonies with no halo = S. aureus

No growth = listeria

Listeria will not grow on Tinsdale

35
Q

What infections does C. perfringens cause

A

Gas gangrene to food poisoning

36
Q

How do you carry out a lactose gelatin medium test?
(4)

A

Ask for a straight wire loop and a lactose gelatin tube

Inoculate test and controls (+ C. perfringens and -C sporogenes and E.Coli)

Incubate at 35 degrees

Refrigerate until chilled and examine for gelatin liquidisation and lactose fermentation

37
Q

What are your controls for the lactose gelatin medium test?
(3)

A

Lactose positive and gelatinase positive = yellow liquid = C. perfringens

Lactose negative and gelatinase positive = red liquid = C. sporogenase

Lactose positive and gelatinase negative = yellow gel = E. Coli

38
Q

How do you carry out an Egg yolk -agar Naglar Test
(7)

A

Ask for Egg yolk agar, C. perfringens antitoxin and C. perfringens control

Split plate in two, label one half C. perfringens type A antitoxin

Swab half of plate with antitoxin A

Allow plate to dry

Inoculate egg yolk agar with a single streak across the plate starting from the side with no antitoxin

Do the same with + control C. perfringens

Incubate at 37 degrees for 48 hours

39
Q

What are your controls for the egg yolk agar/ Naglar reaction?

Only need to put up a positive control as there is not enough room on the plate

A

Lecithinase positive = appearance of opaque, diffuse zone = C. perfrnigens

Lecithinase negative = absence of opaque zone = C. sporogenes

Lipase positive = pearly sheen = C. sporogenes

Lipase negative = no pearly sheen = C. perfringens

Naglar positive = disappearance of opaque zone = C. perfringens

Naglar negative = no change of opaque zone

40
Q

How do you carry out an X/V test for Haemophilus
(6)

A

Ask for X, V, X/V discs, a bijou jar and a Diagnostic Sensitivity Agar (DST) plate

Make a light suspension of the test organism by touching one or more colonies and emulsifying in a reduced volume (avoid picking up chocolate agar)

Lawn the bacterial suspension evenly

Position the three discs on the inoculum

Do the same for your controls

Incubate in 5% CO2 at 35-37 degrees

41
Q

What are you controls for the X,V, X and V factor test

A

Use a H.influenzae control -> needs X and V

42
Q

How do you carry out a growth at different temperatures test for pseudomonas?
(5)

A

Ask for 2 blood/nutrient agar plates

Split plate into 3, test and controls

Zig-zag inoculum of test and controls (+ P. aeruginosa, - P. fluorescence)

Incubate at 42 degrees

Repeat this but reverse controls and incubate at 4 degrees

43
Q

What are your controls for the growth at different temperatures test?

A

+ P. aeruginosa, - P. fluorescence at 42 degrees

+ P. fluorescence and - P. aeruginosa at 4 degrees

44
Q

How do you carry out a cetrimide test
(5)

A

Ask for cetrimide, P. fluorescence and P. aeruginosa

Separate plate into 3 for test and controls

+ control = P. aeruginosa, - control = P. fluorescence

Streak inoculate the test onto the agar

Incubate at 37 degrees

45
Q

What are your controls for the King’s A Medium Test

A

+ control = fluorescent green colonies = P. aeruginosa
- control = non fluorescent colonies = P. fluorescence

46
Q

List the ten components of the biochemical test and the reagents

A

Inoculate a sterile water

  • Peptone (water) indole
  • Buffered glucose Methyl Red
  • Buffered glucose Voges Proskauer Test
  • Peptone sugar dulcitol
  • Lysine decarb base control
  • Lysine decarb test
  • Phenylalanine agar
  • Urea agar
  • MacConkey purity plate
  • Citrate slope
  • Kovacs reagent 8 drops on indole
  • MR reagent on Methyl red
  • VP1 and VP2 reagent on VPT
  • Ferric chloride 3-4 drops for phenylalanine
47
Q

How do you carry out an anaerobic metronidazole susceptibility test for anaerobic GNBs/ any aerobe
(4)

A

Ask for x2 blood agar plates and a metronidazole disc and your controls (Bacteroides fragilis + and pseudomonas fragilis -)

Split your plates into three sections for your controls and test

Inoculate both plates

Add metronidazole discs to your test inoculum - in the middle of secondary inoculum/streaks

48
Q

What are your controls for anaerobic metronidazole susceptibility testing?
(2)

A

+ control = a known anaerobe e.g Bacteroides fragilis (anaerobic growth only)

  • control = a known aerobe e.g. pseudomonas aeruginosa (aerobic growth only)
49
Q

What six antimicrobials make up the MID8 ID Mastring test

A

Erythomycin

Rifampicin

Colistin sulphate

Penicillin

Kanamycin

Vancomycin

50
Q

How do you carry out an MID8 ID Mastring Test
(6)

A

Ask for MID8 mastring (list antimicrobials), blood agar and a McFarland 0.5 standard

Great a dense suspension of organism equivalent to a McFarland 0.5 standard (we didnt do this just lawned)

Lawn inoculum

Using a sterile forceps press the MID mastring onto the plate

Incubate the plate anaerobically at 37 degrees for 48 hours

Do the same for your B.fragilis control

51
Q

What is your control for MID mastring test

A

B. fragilis

52
Q

How do you carry out a disk diffusion antimicrobial test
(7

A

Ask forMueller-Hinton agar

Create inoculum using a 0.5 McFarland turbidity standard

Inoculate plate, rotate 60 dergees 3 times

Place appropriate disks on plate

Press down gently with sterile forceps

Incubate at 37 degrees

Measure zone of inhibition

53
Q

What five disks are used for staphylococci

A

Cefoxitin 30ug

Ciproflaxacin 5 ug

Erythromycin 15ug

Gentamicin 10ug

Penicillin 1 ug

54
Q

What five disks are used for E.Coli

A

Ampicillin ug
Cefotaxime ug
Ceftazidine 10ug
Gentamicin 10ug
Ertapenem 10 ug

55
Q

How do you carry out an MIC

A

Apply lawn inoculum of 0.5 McFarland standard to MH agar

Lawn inoculum, turn 60 degrees three times

Place E test in centre of plate

Incubate and check for growth

56
Q

How do you test for ESBL-mediated resistance?
(4)

A

MH agar

Lawn inoculum

Ceftaxidime and ceftazidime-plus-clavulanic acid discs are added

Measure zone diameters

2.2 Microbiology (60 decks)

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