id tests Flashcards
(88 cards)
1
Q
use of catalase test
A
identify micrococci/staphyloccoci (+ve) from streptococci and enterococci (-ve)
2
Q
carry out catalase test
A
- get colonies from 18-24 hour pure culture using a 10microlitre sterile loop and place on clean cover slide
- 1 drop of 3% hydrogen peroxide on clean glass slide.
- place cover slip on glass slide and observe for immediate bubbling.
- discard in disenfectant
precautions:
do not use blood agae as can give false positive.
3
Q
lysostaphin test and principle
A
identify micrococci (-ve) from staphylocci(+ve)
Lysostaphin cleaves glycine rich pentapeptide cross bridges in staphylococcal cell wall peptidoglycan. cells are rendered to osmotic lysis.
leads to zone of inhibition with Staph aureus as it is lysed. good growth with micrococci
4
Q
carry out lysostphin test
A
- Mueller hinton agar is swabbed with 0.5 McFarland concentration of organism.
- 6mm paper disk impregnated with 10micrograms of lysotaphin are placed on plate.
- incubate at 35-37 for 18-24 hours
5
Q
coagulase test used for and principle
A
id different staphylococci. Staphylococcus aurerus sub. aureus is positive for all coagulase and heat stable endonuclease.
-ability of organism to produce staphylocoagulase enzyme
- staphylocoagulase together with prothrombin form staphylothrombin which activates fibrinogen into fibrin to clot.
6
Q
two types of coagulase
A
- bound coagulase found using the slide method. Cell wall associated and enzymatically converts fibrinogen to fibrin, causing bacterial cells to clump. reffered to as clumping factor.
- free coagulase found using tube procedure. reacts with globulin plasma factor (coagulase releasing factor) to form staphylothrombin–> fibrinogen to fibrin.
7
Q
test procedure of slide coagulase test
A
- one drop of sterile saline )0.85% NaCl) on a clean glass slide.
- gently enulsify a heavy suspension of staphyloccus specimen (18-24 horu pure)
- add 1 drop of latex/plasma in suspension
- observe for immediate formation of macroscopic precipitate.
8
Q
test procedure of tube coagulase test
A
- add 0.5mls of undiluted plasma to 0.5 mls fo nutrient broth 2
- emulsify a large colony into the broth
- rotate tube, do not shake
- incubate at 37 degrees for 4 hrs, observe every 30 mins for clotting. do not shake or agitate tube
- if no clotting is observed, incubate overnight
9
Q
DNase test
A
supplemental test to identify pathogenic staphylococci. also aid in differentiation klebsiella-enterobacter-serratia division of enterobacterales.
ability of organism to produce deoxyribonucleae enzyme that depolymerizes DNA.
Staph aureus is +ve.
10
Q
DNase test procedure
A
stab or line streak with 18-24 hr pure culture on DNase medium. incubate for 18-24 hrs at 37 degrees. flood plate with 0.1M HCl.
area around colony is transparent–> +ve
area around colony is opaque –> -ve
11
Q
DNase test principle
A
enzyme cleaves DNA in the medium surrounding the streak. HCl cleaves DNA that ahsn’t been cleaved yet. presence of enzyme, HCl has nothing to denature, leaving a clear zone around teh streak or stab
12
Q
thermonuclease test
A
staph aureus ability to produce heat stable DNase called thermonuclease. used especially to detect Staph aureues in positive blood culture bottles.
DNA is broken down by thermonuclease, changing toluidine blue to pink
13
Q
carry out thermonuclease test
A
- asepctically inoculate several colonies of isolate into 1ml of brain heart infusion broth
- incubate at 35-37 for 2 hrs
- heat at 100 degrees for 15 minutes and let cool
- cut 6mm diameter wells in plate fo toluidine blue DNase agar using the blunt end o a sterile pipette. (max 12 wells)
- fill each well with cool broth
- incubate at 35-37 for up to 4 hrs and check hourly, do not inverte the plate
positive: pink zone of clearing at the edge of the well. darker blue ring at the outer periphary of the zone. staph aureus.
14
Q
novobiocin test
A
for reliable presumptive id of staph saprophyticus from outer coagulase negative staphs in urine.
staph saprophyticus is the only novobiocin resistant organism isolated from human samples.
15
Q
procedure for novobiocin test
A
0.5 McFarland inoculum is prepared from a pure culture plate 18-24 hrs old and swabbed over a mueller hinton plate. 5microgram novobiocin discs are placed on the plate. the plate is incubated in air for 18-24 hrs at 35-37 degrees.
a zone of 6-12mm shows resistance. –> staph saprophyticus
a zone of 16-27mm indicates sensitivity.
16
Q
differentiate catalase negative species
A
catalase negative are the streptococci and enterococci. swabbed on blood agar to see alpha, beta or no haemolysis.
17
Q
optochin test
A
perfromed on alpha haemolytic organisms. used to differentiate strep pneumoniae from other alpha haemolytic species (viridans sstreptococci). optochin tests the fragility of bacterial cell membranes, and strep pneumoniae is susceptible to it.
full name of optochin is ethylhydrocupreine hydrochloride. optochin has 95% specific sensitivity to strep pneum so other tests should be performed on suspected colonies
18
Q
carry out optochin test
A
- blood agar plate is swbbaed with the suspected pure culture (18-24 hrs old).
- commercially prepared optochin disc
(filter paper impregnated with 0.02 mls of a 1:4000 aqueous dilution and dried at 37 degrees) is placed in the middle of the plate - incubated in CO2 enriched atmosphere (3-5%) at 35-37 degrees for 18-24 hrs.
interpretation:
if strep pneum:
with 6mm disc: 14mm or mroe
with 10mm disc: 16mm or more
varies from 15-30 mm
19
Q
morphology and details of strep pneum against vridans streps
A
strep pneum:
capsulated, diplococci and form draughtsman colonies (this type of colony only formed by capsulated strep pneum)
viridans streps:
non capsulated, short chains and flat regaular colonies
20
Q
bile solubility test
A
used to differentiate between bile soluble (lyse) strep pneumoniae and bile insoluble other alpha haemolytic species. tests the ability of bacterial cells to lyse in the presenc eof bile salts within a specific timeframe at a certain temp.
the bile salts nclude Na deoxycholate and Na taurocholate
21
Q
bile solubility test broth method
A
- heavy suspension of pure culture is prepared in 1ml of sterile saline and seperated into two tubes (test and control).
sodium deoxycholate may precipitate at a pH of 6.5 or less so 0.1M NaOH can be added to adjust the broth culture medium to 7.0pH
- one drop of phneol red in each tube
- 0.5 mls of 10% bile salt solution to tube marked test and 0.5mls of saline added to control tube.
- incubate at 37 degrees or in a water bath for 3 hours (water bath preferred to prevent water loss from broth)
- observe for clearing which should occur within 3 hours
results: clearing or los of turbidity in ‘test’ tube is positive. control remains turbid
22
Q
bile solubility plate method
A
place 1 loopfull of 2% Na deoxycholate directly ona single well isolated colony. keep plate upright, do not invert and incubate at 37 degrees for 30 minutes.
if colony disintegrates, test is potieive.
23
Q
aesculin hydrolysis test
A
used to identify enterococci and group d (+ve) from other streptococci. (strep bovis that forms part of group D is +ve). bile inhibits non group D aesculin hydrolysing streps –> could give false positive
determines the ability to hydrolyze the glycoside aesculin to aesculetin and glucose in the presence of bile.
24
Q
aesculin test procedure
A
bile-aesculin broth or agar is inoculated with pure sample, incubated at 37 degrees for 18-24 hours.
results:
from straw to black colour is positive test–> organism hydrolyses aesculin.
lack fo colour change is negative
25
beta haemolytic species are spearated by Lancefield grouping. mention species in groups
A: Streptococcus pyogenes
B: Streptococcus agalactiae
C: Strep equi, and other zoonotic
D: Strep bovis and enterococcus faecalis
F/G: Streptococcus anginosus. (antigens vary from F,G,A,C)
26
lancefield grouping method
Lancefield grouping testing kits are used.
bacterial cells are mixed with 0.5mls of extraction enzyme. the mixture is incubated at 37 degrees in a water bath for 10 minutes. shake vigorously for 5 minutes. 1 drop of extracted antigen is placed on cards provided with the kit with 1 drop of each grouping latex (different reagent each circle). The extract and latex are mixed using an orange stick, and the card is gently rocked and observed for agglutination.
positive result: visible agglutination/formation fo floccules with latex particles.
negative: milky appearance with no clumping
27
principle behind lancefield grouping test method
the extraction enzyme removes the lancefield group specific material from the bacterial cell walls, leaving them in soluiton.
drops of the extract react with the individual reagents containing latex particles coated with antibodies to lancefield groups A,B....
agglutination is seen in the well corresponding to the extracted antigen.
agglutination is observed between enzymatically extarcted antigens and antibody coated latex particels
28
PYR test principle
detect presence of L-pyrrolidonyl arylamidase which is produced by organisms. this enzyme is used for the conversion of L-pyrrolindonyl beta naphtylamide into L-pyrrolidone and free beta naphytlamine. beta naphytlamine is the substrate for the PYR reagant which produces a red colour.
In summary: the eznyme creates the subtrate that upon reaction with the PYR reagent creates a red colour
presumptive test for group A beta haemolytic streps and group D enterococci.
more sensitive than salt tolerance test and bacitracin test
mainly enterococci vs strep bovis
29
how is PYR test carried out
incoulate colony with PYR broth and incubate at 35 for 4 hours, and add drop fo PYr reagent to observe red colour.
red colour is presence of enzyme --> enterococcus
30
salt tolerance test
check ability of organism to grow in variable amounts of NaCl. .
distinguish between enterococcus and other group D streptococci.
31
hwo to carry out salt tolerance test
brain heart infusion broth or pepton water broth is used. the salt content is increased to 6.5%. some media can incorporate an indicator. 3-5mls of the medium are sterilized at 121 for 15 minutes.
2/3 colonies are inoculated into the broth and incubatd for 18-24 hours at 35-37 degrees.
positive: bacterial growth with or without colour change of indictoar.
32
precautions with salt tolerance test
- do not inoculate heaviliy to avoid false positive result as you identify remnants as growth
- swirl tube well before interpreting to avoid false negative.
- group b, a and aerococci may give fals positive.
33
identifying between enterecoccus group d and tstreptococcus bovis
enterococcus is positive results for all aesculin (colour change to black), pyr (change to red) and salt tolerance (grows)
strep bovis gives a positive result for aesculin but neative for pyr and salt tolerance
34
hippurate hydrolysis test
detect ability of bacteria to produce hippuricase which hydrolyses hippurate into glycine and benzoic acid. glycine is detected by oxidation with ninhydrin reagnet reseulting in a deep purple colour
used as a confirmatory test
35
which species hippurate test identifies
strep agalactiae from other beta haemolytic ones, listeria monocytogenes, campylobacter jejuni and campylobacter strains, gardnerella vaginalis
they are all psoitive
36
carry out hippurate test
- 0.1 mls of distilled water to 12x75mm test tube.
- make a heavy suspension with the test organism.
- add hippurate disc in the mixture and cap
- incubate for 2 hours at 35 degrees (water bath is preffered)
- add 0.2 mls of ninhydrin reagent and re incubate for 15 to 30 minutes.
-
negtaive --> colourless or slightly yello-pink colour
positive--> deep purple colour
37
limitations of the hippurtae test
if incubation with ninhydrin reagent lasts more than 30 minute --> false positive result.
ninhydrin slution deteriorates within 6 months
insufficient inoculum may result in erroneuous results
38
what are X and V factors
growth factors that may be required by haemophilus species to grwo. qualitative procedure for isolation and differnetiation fo haemophilus species.
only haemophilus influenzae, haemolyticus and aegyptius needs both factors
39
difference bteween X and V factors
the X factro si the heme portion of Hb necessary for the synthesis of respiratory enzyms and is heat stable
The V factor is coenzyme NAD which is heat labile
40
X and V factor test procedure (w/o strips)
- chocolate agar medium provides X factor and yeastrel agar medium provides neither factor
-both swabbed in the middle with Haemophilus species
- centre is stabbed with pure culture of Staph aureus to procide V factor
-plates incubated at 37 degrees for 24 hours in 3-5% of CO2
growth on both: X and V or V only
no growth on V: only X
41
test procedure for X and V factros with strips
culture media used is deficient of both factors: mueler hinton, trypticase soy medium, or brain heart infusion medium
- swab on surface
- add strips impregnated with X and another with V
- incubate at 37 degrees for 24 hrs in 3-5% CO2
-observe for satellitism (coloniesof haemophilus are larger near factor and decrease in size away from it)
42
oxidase test
detects presence of c components of the cytochrome oxidase system used at the end of aerobc respiration. change from clear to coloured when converted from the reduced to oxdised state.
used to distinguish between pseudomonadaceae and enterobacterales (both italics)
- pseud are aerobic so psoitive and give purple colour, neisseria are also positive
- enterobacterales are facultitevly anaerobic so negative.
with oxygen, reduced cytochromes become oxidised with with the reagent form a coloured compound.
43
carry out oxidase test
1) few crystals of Kovac's reagent (freshly prepared) is added to 10mls fo sterile distille water
2) few drops are placed on filter paper
3) glass or plastic rod is used to emulsify a colony on the filter paper (colony from nutrient agar ideally)
positive is a purple formation within 10 seconds
44
precaustion for oxidase tets
do not use blood agar for colonies as picking up blood could casue false potitive reuslt
45
use fo OF test
identify between aerobic and anaeroic, mainly used to differentiate between gram negative bacilli (enteric pathogens). even help in differntiation fo staphs from microcci
fermentative: anaerobic usually facultative, both turn yellow
oxidative: usually obligate aerobes, open turns yellow
alkaline: cannot breakdown carb aerobically or anaerobcially form blue colour
46
test procedure of OF
two tubes of OF basal medium with 10 % dextrose are stab inoculated with a suspension of the organism in sterile saline. one tube add 1cm of paraffin oil (anaerobic)
incubate at 37 for 18-24 hrs
47
results of OF and oxidase of
-enterobacterales
- pseudomonas
- acinetobacter
-alcaligenes
-aeromonas
enterobacter: fermentative and negative
pseudo: oxdiative and positive
acinetobacter: oxidative and negative
alcaligenes: oxidative/alkaline and positive
aeromonas: fermentative and positive
48
urease test
determine ability to produce urease and break down urea into 2 molecules of ammoina --> alkaline.
- used to differntiate proteus (positive and rapid result) from other enterobacterales
- differentaite between enteric bacteria and salmonellae
49
carry out urease test
heavy inoculate christensen urea agar slants in 2 ml mCCartney bottles and incubate at 37 for 18-24 hours.
+ve pink colour
-ve amber colour
50
indole test
ability to produce indole from the oxidation of tryptophan (using tryptophanase). formation of red colour after benzaldehyde reagent.
-differentiates between genera within family of enterobacterales.
E. coli +ve
E. vulneris -ve
Salmonella spp. +ve
Klebsiella -ve
Protues vulgaris +ve
Proteus mirabilis -ve
Yersinia pestis -ve
- aids in differentiation with other test
- spot indol tets to subdivide H. influenzae into subtypes (called biotypes)
51
carry otu indole test
heavy inoculate 1%tryptophan or peptone broth. incubate for 24-28 hours at 37 degrees (incubate aerobically as low O2 tension decreases indole production).
0.5 mls of Kovac reagent is added and gently swirl the tube.
red layer forming at the top of liquid layer --> positive
yellow/amber colour top of liquid layer--> negative
52
ONPG (beta galactosidase) test
test for presence of beta galactosidase, which breaks down ONPG (O- nitrophenyl-beta-D-galactopyranoside) into galactose and o-nitrophenol which has a yellow colour to show hydrolysis. tehrefore yellow--> positive test.
-differentiate between enterobacterales (E.coli +ve and Proteus mirabilis is -ve)
- differntiate neisseria lactamica (+ve) from other neisseria (-ve)
53
carry out ONPG test
few discrete bacterial colonies from non selective media are inoculated in tubes containing ONPG and incubated at 35-37 degrees for up to 24 hours.
they are examined for a yellow colour after 4 hours up to 24 hours.
+ve turns yellow
-ve solution is colourless or pale yelllow
54
types of indole tests
- spot indole test used for rapid indole production, well siolated colony needed
- tube method to identify weak indole producing organisms
- tryptophan or peptone water medium
55
precautions with ONPG test
- discard media if it looks yellow prior to incubation
- do not use organisms having a yellow pgment
- ONPG solution should eb correctly buffered to present false results.
56
Methyl red test
used to detect a very large production of acids, produced from the breakdown of carbs. (lactic, acetic, formic).
Differentite between members of enterobacterales as they produce differnet amounts of acids.
MR is yellow at 6.0 and red at 4.4
methyl red positive organisms overcame the pH buffering system and maitain a low pH on prolonged incubation
57
carry out methyl red test
- uses MR/VP broth (methyl red/ voges proskauer broth). has a pH of 6.9 and amde of peptones, glucose, dipotassium phosphate and distilled water. methyl red reagent is 95% in ethanol and distilled water.
1) inoculate the broth with 18-24 hours culture
2)incubate at 35-37 degrees for 48-72 hours(not less than 48).
3) add 5 drops of methyl red reagent and interpret results.
+ve -> red colour--> E.coli
-ve --> yellow/orange --> Enterobacter aerogenes
58
voges- proskauer test
detect the production of acetoin from pyruvic acid, in breakdown of glucose. production of a red-pink complex in the presenc eof O2 and 40 % KOH.
detect acetoin an intermediate product int eh butene glycol pathway of glucose fermentation.
glucose is broken down to pyruvate, which is made into acetoin , in the presence of KOH acetoin is oxidised to diacetyl. diacetly reacts with arginine and alpha naphtol (colour intensifier) to produce a red colour.
used to differentiate klebsiella-enterobacter-hafnia-serratia group from others as they are positive. differentiates between enteric rods.
59
positive and negative results for voges proskauer test
+ve : enterobacter (all, eg. aerogenes) listeria, klebsiella, hafnia alvei, serratia marcescens.
-ve: E.coli, shigella, yersinia
60
carry out voges proskauer test
1) inoculate MR/VP broth with culture and incubate for 24hrs at 35-37 degrees.
2) to 1ml of broth, 0.6mls of 5% alpha naphtol (colour intensifier) and 0.2 mls of 40% KOH(oxidising agent) are added.
3) they are mixed well and and leave undistirbed for 10-15 minutes
4) observe results after 10-15
preucation: do not leave more than 1 hour bc reagents may produce a copper like colour giving false potiigve.
61
citrate test
determine the capability fo the organism to utilize citrate as a sole sorurce of carbon for ematbolisma dn growth. If citrat eis used it is detected by alkaline by products.
the medium used is simmons citrate medium it is a solid medium slant, with Na citrate as its sole source of C and ammonium phosphate as the sol esource of N.
Klebsiella pneumoniae is citrate positive
62
carry otu citrate test
1) incoulate the medium in a single streak with colonies 24-48 hrs old.
2) incbuate at 35-37 degrees for 24-48 hours. no tmore than 48.
interpret results:
+ve is green chnaging to deep blue (Proteus rettgeri
-ve remains as a green colour, growth may occur still by those that do not assimilate N. . eg Morganella morganii
63
rapid citrate utilisation test
used to test the ability of anorganism to use citrate in citrated blood to form clot.
1) brain heart infusion broth + CaCl2+ NaCl is sterilized at 121 for 5 minutes
2)15mls of cutarted blood are added to medium
3)aliquoted into 1 ml volumes and heavily inoculated with 4-5 colonies (if 2-3mm) that are 18 hours old.
4) incubate at 35-37 for 1-6.5 hours
presence fo a firm clor it a positive result
64
decarboxylase test
measure the ability of an organism to decarboxyalte an amino acid into an amine ( +alkalinity) by the products' effect on pH and colour change. used primarily with gram negative rods
lysine and ornithine decarboxylate directly and arginine is first converted to citrulline.
65
carry out decarboxylase test
use moeller decarboxylase broth base with a pH of 6. Has peptone, beef extract, glucose, bromcresol purple indicator (yellow 5.2 and purple at 6.8), cresol red indictaor (yellow 7.2 and red at 8.3), pyridoxal phosphate enzyme
1) 4 tubes are prepared: 1 control with just broth no amino acids, other 3 with rboth and 1% of L-lysine another L-Ornithine and another L-arginine.
2)incoulate broths heavily
3) overlay all tubes with thin layer of sterile paraffin oil (glucose is fermented, prevents oxidative degredation and O2 in medium is consumed by organism)
4) incubate at 35-37 degrees for 24-96 hours
+ve blue purple colour
control shoudl eb yellow as pH was lowered to activate decarboxyalse enzymes
66
gelatine liquefaction test
ability to produce gelatinases and break it down. Proteins are brokwn to polypeptides, frutehr browkne to amino acids
differentiate between:
--> Proteus hauseri (+ve) vs Providencia stuartii (-ve)
--> Staph aureus (+ve) vs Staph epidermidis (-ve)
--> Nocardia brasiliensis (+ve) vs Nocarida asteroides (-ve)
67
carry out gelatin liquefaction test
use Kohn Gelatine Medium with denatured gelatine and charcoal discs to visibly show that gelatine is liquified as it is colourless.
(other media that can be used are Nutrient Gelatine Plate medium or Thioglycolate Gelatine medium)
inoculate medium (in peptone water) with heavy inoculum. incubate for 18-24 hours.
+ve result: liquefaction of gelatin disc with charcoal depositing on the bottom
run control tube to check growth if no liquefaction
68
carbohydrate fermentation
determine ability to break down sugar incorpoated in the medium. can produce acid or acid and gas
results with some sugars
-enterobacterales all ferment glucose
- Xylose: proteus +ve (acid), Provedencia -ve
- mannitol: Staph aureus +ve and Staph epid -ve (on MSA)
- (On CTA cystine, trypticase medium) Nesseria men. glucose and maltose +ve
Nesseria gonor. glucose + maltose -ve.
Lactose
E.coli +ve and Staph aureus +ve
Proteus mirabilis and salmonella -ve
-
69
carry out carb fermentation tets
broth
-peptone water
- beef extract optional
- NaCl
-distilled water
-phenol red (acid yellow 6.8) (alkaline red 8.4)
and 10% carb solution
inoculate broth with suspension in peptone water or sterile saline. incubate for 18-24 horus, interpret reaction colour change.
for gas detection insert inverted Durham tube before sterilizing broth to check for gas bubble.
70
precaution when testing carb fermentation of neisseria on CTA medium
neisseria requires 7-10% of CO2 to grow. plaed in a CO2 incubator and caps are left a bit open. this could lead to medium becoming yellow due to CO2 false positive. therefore instead, remove from incubator withou closing cap and leave on bench for 20-30 minutes then carry out interpretaion.
71
nagler test
identify organisms taht produce lechitinase, a phospholipase enzyme. the alpha toxin of clos prfringens has phospholipase activity so allows for differentiation ebtween clos. perfringens and other clostridia that produce lechitinase,, by neutralization of lechitin c activity by the antitoxin.
lechitin is found in egg yolks, lechitinase brekas it down into phosphorylcholine and insolubel diglyceride that froms the opaque pp around colonies.
+ve--> zone of opacity in antitoxin free zone but not in otehr half due to neutralization::: Clostridium perfringens
-ve--> zone of opacity on both sides or none at all.
72
carry out nagler test
1) mark a plate of egg yolk medium in half.
2)inoculate half the plate with 60 microlitres of clostrdium perfringens type A antitoxin.
3) spread with 'hockey stick' spreader or 10 microlitre loop.
4) allow to absorb and dry completely
5) streak organism from antitoxin free to antitoxin side.
6) incubate anaerobically at 35-37 degrees for 24-48 hours
control irganisms done in same way
73
KOH test
used as a complement test with the gram stain to identify gram negative from gram positive organisms. bacillus and clostridium species may lsoe their integrity during a gram stain and be considered as gram negative. this test is based on chemical differences of the gram negative adn positive cell wall.
In KOH, gram neg cell wall is broken down releasing chromoomal material, making the bacterial suspension thick and tringy, nothing happens to gram psoitive.
(older cultures may still break down - more than 48 rs)
reagent must be freshly prepared.
74
carry out KOH test
1) 1 drop 3% KOH on lean mciroscope slide
2) emulsify a few coloinies to make a dense susension
3) stir continuously for 60 seconds then gently pull away the loop away from the suspension
4)If positive, a string of suspension will follow the loop whne it is raised (chromosomal material) - within the first 30 seconds
75
triple sugar iron agar
differentiate enterobacterales by 3 sugar fermentations: sucrose, lactsoe and glucose. smei solid medium with butt and slant. subculture can be made from salmonella chromogenic agar, macckoney 3.
76
carry out tests on triple sugar iron agar
incoulate TSI with 18 hour old culture, smear the slope and stab the butt, incubate at 37 degrees. in parallel incoulate a urea broth as well (when differentiating slamonella from proteus)
after 5 hours examine urea broth:
urea is positive--> proteus contamination so throw away TSI
if negative examine TSI after 18 hours and 48 horus
77
observe motility
either by stabbing solid medium or under microscope
solid medium made f beef extract, peptone, sodium chloride, 4g/L agarnd distilled water placed in 14ml McCartney bottle not plate. medium is stabbed and incubated for 18-24 hrs at 37 degrees. grwoth away from stab line- FARING- shows motility. (growth along line is not motility). peritrichous may even trun medium opaque.
heavy inoculate a tryptone or peptone broth and incubate for 4-6 hrs at 37 degrees. place loopful onto a clean microscope slideand cover with a cover slipand view under high dry magnification. motile bacteria are observed from browniam motion. to keep anaerobes alive, the inoculated broth is placed in a capillary tube closed each side with clay.
78
types of motility from different types of flagella
monotrichous--> darting
amphitrichous--> tumbling
peritrichous --> very fast.
79
capsule india ink stain
negative stainig, staining fo the background not the bacteria, capsules displace the dye and are visible as halos around bacteria.
demonstates capsule in streptococcus pneumoniae, cryptococcus enofromans.
80
carry out idnia ink capsule test
drop of india ink on a clean glass slide. add one drop of either specimen, liquid culture or colony, near the ink drop then mix. place a 22x40/50 cover slip. press down through blotting paper for a thin film.
positive: organism appear refractile, with clear zone against a dark background
negative: no clear zone observed
81
mc farland standards
created with BaCl2 and H2SO4 mixtures to create barium sulphate standards. opacity translated into CFU
82
sealents used
mineral oil or paraffin. 100mls with 1ml of distilled water. sterilize with ot air oven at 160-170 dgrees fro 1 hour
vaspar, mixture of 1:1 petroleum jelly and paraffin. sterilize in 50ml parts in dry oven at 100 degrees to drive off water
83
inoculum for susceptibility testing
prepared from 4-6 hours colonies incubated in broth, or young colonies incubated overnight on plate. should have turbibity of 0.5 McFarland
84
broth dilution techinque for susceptibility testing
1) perform doubling dilution of the antibiotic in liquid growth medium- Mueller Hinton
2) add a lwo concentrtion of test bacteria to each dilution (0.5 McFarland)
3) add controls: -antibiotic - organism
+ antibiotic -organism
-antibiotic +organism
4) incubate ofr 18 to 24
For Minimum inhibitory concentration: the first broth dilution with concentration of antibiotic in mg/L that shows no visible growth
for minimum bactericidial concentration: 5 broth dilution from the one that showed no visible growth are plated to observe growth. incubate. Mbc is the plate that has absolutely no growth on it.
85
stokes technique for susceptibility testing
dried surface of nuller hinton plate is divided into 3 parts. the upper and lower parts are inoculated by streaking the control strains. the test strains of the same type are inoculated in theh centrer. inocula on plate left to dry and disc is placed aseptically between the strains.
sensitive: zone of inhibition is not more than 3mm smaller, or equal to the control.
intermediate: zoen is more than 2mm but still more than 3mm smaller than the control
resistant: no zone of inhibition or smaller than 2mm
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kirby bauer technique
swab mueller hinton agar or solid medium, make sure dry, for confluent growth, aseptically place antimicrobial discs in a circular fashion (equidistant as much as possible and not more than 9 discs on 90mm plate).
incubate at 35 to 37 18-24 hrs
2 measurement sof zone of inhibtiion taken and shape is also considered.
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antimicrobial inetractions of kirby bauer technique: shapes you can get
synergism: bridging of zone of inhibition. combined effect. found with aminoglycosides and penicillins
antagonism: antimicrobial will negatively affect the other antimicrobial on the side of the straight line (penicillins and bacteriostatic drugs)
autonomy: seperate zones of inhibition, circles, do not affect eachother,
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gradient method
uses an antibiotic strip with a an exponential gradient of the antibiotic along it. the rate of diffusion is directly proprotional to the concentration at a specific locus. will form the shape of an ellipse hance E test, the diffusion will show the zone of inhibition.
used to monitor MIC values of highly resistant organisms.
