Precautions of Tests

Question 1

what are some precautions of the citrate test

Answer 1

keep inoculum light because even dead organs can be source of carbon - false pos

loose caps - if too tight false neg

incubated for 24 hours

flame the wire before inoculating to avoid carryover of glucose - false positive

-some orgs that grow on citrate dont produce a color - still pos citrate

DO THIS TUBE FIRST

Question 2

O/F PRECAUTIONS

Answer 2

loose caps to help utilize proteins and get alkaline conditions - if too tight false neg

can have slow growers incubate long

media is general purpose
mineral oil can be acidic - result error

-dont use staph from mannitol salt plate since O/F media for staph has mannitol and glucose

any yellow shade is pos

-inoculum should NOT be from lactose
-heavy inoculum is required
-dont test with yellow pigmented orgs
-dont use orgs that grown on glucose - glucose inhibits or slows B galact production
-do not report before 24hr

Question 3

TSI PRECAUTIONS

Answer 3

test invalid if the butt of the agar is not stabbed or a pure colony is not used
-dont use loop the crack could look like gas production
-If caps too tight, slant will not revert to red = a false acid result (leave caps loose)

If read before 18h a false positive of A/A may occur
If read after 24h a false NA/NA may occur due to all the sugars being used up and only peptones being left to use

Question 4

MR PRECAUTIONS

Answer 4

-should not be read before 48 hours some organisms will not have produced enough products from the fermentation of glucose but we have to read at 24 hours because of school

• Don’t use heavy inoculum, since this may inhibit proper bacterial growth within the medium
• An orange endpoint may be due to excessive broth volume

Question 5

VP PRECAUTIONS

Answer 5

Organisms that are MR positive are generally VP negative and vice versa.

Reversing the order of adding α naphthol and KOH may give a weak positive or false-negative result.

Do not exceed the recommended volume of 40% KOH. - masks a weak pos

Reaction is enhanced if the tube is mixed to expose the contents to oxygen helps promote reaction acetoin to diacety

Question 6

DECARBOXYLASE PRECAUTIONS

Answer 6

Must include a control tube even if many tubes are tested only one control is needed

-The tubes (both control and test) must be sealed with sufficient mineral oil to prevent the uptake of oxygen. Decarboxylase enzymes require an acidic environment to activate

Medium must be inoculated with sufficient organisms. no turbidity with less acid to change the indicator could show false positive

Question 7

PPD PRECAUTIONS

Answer 7

-green color reaction of a positive test fades rapidly
-results must be interpreted within 5 mins
-agitate the tube slightly

Question 8

SIM PRECAUTIONS

Answer 8

-inoculate from solid media because broth will delay growth
-remove the needle in a line so you dont cause a false pos for motility
-keep caps loose
-Motility and H2S results must be interpreted prior to addition of Kovacs Reagent

-only test PURE culture
-dont test from media with glucose

-if you are unsure of the motility result you can perform a WP or DF motility
-if H2 S produced and the blackening is throughout the media then it is assumed
the organism is also motile

Question 9

NITRATE CONSIDERATIONS

Answer 9

-Reagents A and B are both unstable. Note the reaction colour immediately as it fades over time.

-With fermenting organisms hydrogen gas instead of N2 gas may be present as a bubble therefore continue to add reagents

-Excess zinc dust has been reported to cause false-positive

Question 10

UREA PRECAUTIONS

Answer 10

-Prolonged incubation may cause alkaline reaction in the medium and give false positive result
-do not use inoculum from a broth suspension
-loose caps
-Urea is light sensitive and can undergo auto-hydrolysis put in the back of the incubator

Question 11

what does the vitek need

Answer 11

-identifies most Enterobacteriaceae and GN 8-10 hrs
-* Vitek 2 Antimicrobial Susceptibility Tests (AST) are for aerobic gram negative bacilli- Staph,

saline to mix sample in a suspension
tubes and caps
densichek - 0.5-0.63 density
pipettes 0- color coded GN is red GP is purple

the ID cards need to be warmed to room temp each has a barcode and put in a casette
-ID CARD BEFRORE AST

Question 12

DensiCHEK

Answer 12

photometer which provides values in McFarland units
-measure absorbance of particular wavelengths
-concentration is proportional to intensity of solution color
-the darker it is the higher the mcfarland reading
always zero before use and calibrate between standards

Question 13

how can vitek be contaminated

Answer 13

• Improper handling of the specimen
• Testing from mixed culture plates
• Using contaminated saline
• Improper sterilization of a saline dispenser
• Always make a Blood purity plate from your ID suspension

Question 14

CONSIDERATIONS TO VERIFYING VITEK
RESULTS

Answer 14

-always check the purity plate
-if mixed DONT RELEASE RESULTS
-Each ID & AST results must be examined for typical results for
that organism, cmi , ensure the susceptibility pattern is followed if it is very resistant then something is wrong
-there are 6 ID confidence levels for vitek 2 - Excellent 96-99, very good 93-95, good, 89 acceptable 85, low or unidentified
-only over 90 % is accepted anything check the plate if mixed

There are 4 Vitek Ast level with Green light being the best and consistent and Purple being - analysis not preformed

Question 15

SLASHLINE in vitek

Answer 15

Certain species may belong to a slashline (mixed) taxa identification.
* This occurs when the biopattern is the same for the taxa listed.
if found do the quickest test or chose organism with highest %

Question 16

BETA LACTAMASE

Answer 16

*Staphylococci spp. can make an enzyme that inactivates any
Penicillin class of antibiotics
*VITEK will not interpret the AST results for any penicillin drugs
unless you input the Beta lactamase result as a off line test

Question 17

limitation of CATALASE

Answer 17

• Blood cells contain catalase and will cause false positive reaction if agar with blood is picked with the organism.
• 3% hydrogen peroxide reagent is unstable and must be fairly
  fresh.
• Growth for catalase testing must be taken from an 18-24 hour
  culture. Organisms lose their catalase activity with age, resulting in a false-negative reaction.
  *enterococci) produce a peroxidase that slowly catalyzes the breakdown of H2O2, and the test may appear weakly positive.
• Platinum wires cause false positive results

Question 18

limitation of PYR

Answer 18

• A false-negative test can result if the disk is too moist.
• False-negative tests can result if you take colonies from selective media or tube biochemical agars
• Escherichia coli and indole-positive Proteus spp. obtained from media containing a high Tryptophan content may yield a blue-green color development. This is a negative result.

Question 19

limitation of OPTOCHIN

Answer 19

*Optochin susceptibility is a presumptive test only. DO MORE
-Other strains of alpha-hemolytic streptococci may show a slight susceptibility to optochin
Occasional strains of optochin resistant S. pneumoniae
have been found - a confirmatory test should be performed eg the bile solubility

• S. pneumoniae isolates should be incubated in a CO2 enriched
  environment, as some isolates will grow poorly or not at all
  aerobically

Question 20

OXIDASE TEST imitations:

Answer 20

• Selective media or media containing glucose like MacConkey
  should not be used – fermentation inhibits oxidase activity
  leading to false-negative results. The indicators in the media may also result in false-negative results.
• Reagent can auto-oxidize, and false positive result may be obtained if you wait too long to read the results read within 30 seconds.
• Use platinum loop, chromium loop, or applicator stick –others cause false positives
  Reagent (if made in the lab)
• Should be made fresh daily

Question 21

SPOT INDOLE Limitations:

Answer 21

• Test only colonies cultured on media without glucose, as glucose inhibits indole production.
• Don’t use colonies from MAC, because the color of lactose-fermenting colonies on this medium can interfere with test interpretation and also indicators in these media may cause a false positive result.
• Mueller Hinton Agar should not be used for this test because
  tryptophan is destroyed during acid hydrolysis of casein.
• Certain strains of Proteus vulgaris, Providencia and Aeromonas spp. will give false negative reaction with the
  spot indole test.

Question 22

DNASE Limitations: *******

Answer 22

Some MRSA strains do not give positive DNase test result
* Some strains of the coagulase-negative staphylococci such as
Staphylococcus capitis may give weak reactions.
* It is easiest to read the results if you hold the plate against a white paper

Question 23

BILE SOLUBILITY LIMITATION

Answer 23

Limitations:
* Must use an 18 to 24-hour growth of organism on 5% sheep blood agar or CNA;
-circle the colones from 18-24 hr growth to be tested so you can find it after testing
-incubate plate with lid facing upwards 35 in O2
- Do not move the plate around or colonies will wash away
(yielding a false-positive result)

Question 24

Maldi

Answer 24

has 3 calibration spots
-thin even layer of colony ,overly thick or uneven spots often yield no identification, too thin the signal
generated may be inadequate
MUST COVER WHOLE CIRCLE
-ADD 1uL OF matrix and let air dry - add by capillary action
-Organism must be 18-24 hours old