Day 1: Question 2 - Inoculating Tests Flashcards
How do you carry out a DN’ase test?
(8)
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)
What are your controls for the staph DN’ase test?
Positive clear distinct zone of clearance = S. aureus
Negative cloudy = S. epidermidis or S. saprophyticus
What are your controls for mannitol salt agar?
positive yellow colonies = S. aureus
negative pink/red colonies = S. epidermidis or S. saprophyticus
What are your controls for SAIDE agar?
Positive pink colonies = S. aureus
Negative non-coloured colonies = S. epidermidis or S. saprophyticus
How do you carry out a Staphaurex Plus test
(6)
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
What are your controls for the Staphaurex test
Positive agglutinating control = S. aureus
Negative = coag neg staph = S. epidermidis or S. saprophyticus
How do you test for novobiocin resistance?
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
What are your controls for the novobiocin resistance test?
Positive resistant = S. saprophyticus
Negative susceptibility = S. Epidermidis/ S. aureus
How do you carry out an optochin susceptibility test
(7)
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
What are your controls for optochin susceptibility?
Positive susceptibility = Streptococcus pneumoniae
Negative resistance = Streptococcus viridans
How do you prepare your bacteria for the Lancefield agglutination test?
(4)
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
How do you carry out the Lancefield grouping test
(6)
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
How do you carry out a Bacitracin susceptibility test?
(6)
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
What are your controls for the bacitracin test?
Positive susceptibility = S. pyogenes
Negative resistance = any other staph or Enterococcus e.g. E. faecium
What are your controls for MacConkey agar?
Positive pink colonies = Enterococcus
Negative yellow colonies = gamma haemolytic strep e.g. Strep bovis
How should you carry out a bile susceptibility test?
Ask for a bile aesculin agar plate, E.faecalis + control, Strep pyogenes negative control
Spot inoculate
Incubate at 45 degrees !!!!
What are your controls for bile aesculin agar test?
Positive blackening of medium = enterococcus e.g. E. faecium
Negative no colour change = S. pyogenes
What four sugars are used in the TAXO sugar utilisation test?
Glucose
Maltose
Sucrose
Lactose
How do you carry out a TAXO sugar utilisation test
(6)
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
What should you use as your control for TAXO sugar test?
N. lactamica
How do you carry out the catarrhalis disk test
(3)
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
What are your controls for catarrhalis disk test?
Positive = blue-green colour development = M. catarrhalis
Negative = no colour development within 2 mins = Neisseria
What feature of catarrhalis can be seen from a colony?
Hockey-puck colonies
What are you controls for Moraxella DN’ase test?
Positive = zone of clearance = M. Catarrhalis
Negative = cloudy = Neisseria
How do you carry out a PEMBA test?
Ask for PEMBA, a B. cereus control and a B. subtilis control
Streak inoculate
Incubate at 37 degrees Celsius
What are your controls for PEMBA?
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
How do you carry out the motility test
(6)
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
What are your controls for motility test?
Positive control = motile = any bacillus other than anthacis
Negative control = non motile = B. anthracis is the only non motile Bacillus
How do you carry out the Trehalose and Urease biochemical tests
(4)
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
What is the best way of inoculating the trehalose agar?
(2)
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
What is the best way of inoculating the urea slope?
Zig-zag across the surface of the agar and stab the slope with the loop
What are your controls for the trehalose and urease tests?
Positive trehalose = yellow = C. ulcerans
Negative trehalose = pink = C. diptheria
Positive urease = pink = C. ulcerans
Negative urease = yellow = C. diptheria
How do you carry out a Tinsdale test
Ask for a Tinsdale agar, C. diphtheriae and a diphtheroid such as haemophilus, neisseria, staphylococcus and strep
Incubate at 37
What are your controls for the Tinsdale test?
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
What infections does C. perfringens cause
Gas gangrene to food poisoning
How do you carry out a lactose gelatin medium test?
(4)
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
What are your controls for the lactose gelatin medium test?
(3)
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
How do you carry out an Egg yolk -agar Naglar Test
(7)
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
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
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
How do you carry out an X/V test for Haemophilus
(6)
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
What are you controls for the X,V, X and V factor test
Use a H.influenzae control -> needs X and V
How do you carry out a growth at different temperatures test for pseudomonas?
(5)
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
What are your controls for the growth at different temperatures test?
+ P. aeruginosa, - P. fluorescence at 42 degrees
+ P. fluorescence and - P. aeruginosa at 4 degrees
How do you carry out a cetrimide test
(5)
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
What are your controls for the King’s A Medium Test
+ control = fluorescent green colonies = P. aeruginosa
- control = non fluorescent colonies = P. fluorescence
List the ten components of the biochemical test and the reagents
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
How do you carry out an anaerobic metronidazole susceptibility test for anaerobic GNBs/ any aerobe
(4)
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
What are your controls for anaerobic metronidazole susceptibility testing?
(2)
+ control = a known anaerobe e.g Bacteroides fragilis (anaerobic growth only)
- control = a known aerobe e.g. pseudomonas aeruginosa (aerobic growth only)
What six antimicrobials make up the MID8 ID Mastring test
Erythomycin
Rifampicin
Colistin sulphate
Penicillin
Kanamycin
Vancomycin
How do you carry out an MID8 ID Mastring Test
(6)
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
What is your control for MID mastring test
B. fragilis
How do you carry out a disk diffusion antimicrobial test
(7
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
What five disks are used for staphylococci
Cefoxitin 30ug
Ciproflaxacin 5 ug
Erythromycin 15ug
Gentamicin 10ug
Penicillin 1 ug
What five disks are used for E.Coli
Ampicillin ug
Cefotaxime ug
Ceftazidine 10ug
Gentamicin 10ug
Ertapenem 10 ug
How do you carry out an MIC
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
How do you test for ESBL-mediated resistance?
(4)
MH agar
Lawn inoculum
Ceftaxidime and ceftazidime-plus-clavulanic acid discs are added
Measure zone diameters
