Upload
phungdieu
View
216
Download
0
Embed Size (px)
Citation preview
DR. R P GOVT.MEDICAL COLLEGE, KANGRA AT TANDA DEPARTMENT OF MICROBIOLOGY BACTERIOLOGY LABORATORY STANDARD OPERATIVE PROCEDURES MANUAL(SOPM) GENERAL PRINCIPLES a) Standardized procedures to be followed.
b) Disinfection of working bench at the start of work and end of day. In case of Spillages – cover with 0.5-1% Hypochlorite or bleach or 3% Lysol for 10 minutes then wipe off with absorbant paper and discard in discard jar.
c) BMW must be discarded in colour coded buckets/ bags as per guidelines . d) Hand Washing with soap and water before leaving the laboratory . e) Proper entry and labelling of samples after receiving. and timely dispatch of reports.
Investigations offered in the Bacteriology Section: DirectExamination Stain- - Gram’s stain - Albert stain
- Ziehl Neelsen ( ZN) - Methylene blue - Giemsa stain
- India Ink Hanging drop preparation: for Motility of Vibrio cholerae
1
Culture Isolation- Blood- CSF- Sputum- Urine- Throat swab- BAL- Lung aspirate- Pus- Joint fluid- Stool- Bile- Semen- Catheter tips etc- Blood bags for sterility testing
Antibiotics Susceptibility testing - Disc diffusion method( Kirby Bauer)
MRSA screening of clinical isolates ESBL screening of clinical isolates A. General Instructions for bacteriological examination;
The clinical state of the patient will not be reflected by the result of the laboratory investigations despite correct laboratory test unless the specimen is in optimal condition required for analysis.
1) All samples to be properly stoppered or plugged. There should be No leakage of samples .
2
2) Proper handling of the samples. 3) Preferably to be collected before administration of antibiotics . 4) Prevention of contamination of specimen with externally present
organisms or normal flora
5) Collect the specimen at the appropriate phase of the disease and adequate in quantity for the desired
tests to be performed . 6) Collect sample in sterile appropriate containers as follows :- i) Blood - Blood culture bottles with screw cap / Bactec culture vials . ii) Urine - Sterile, wide mouth container iii) Sputum - Sterile, wide mouth container iv) CSF, pleural, peritoneal fluid or other fluid - sterile container. v) Faeces - clean wide mouth container vi) Swabs from throat other wound/discharge and high vaginal/ cervical swabs - sterile swabs in test tube properly plugged. Do not break off the end of the swab stick. vii) Tissues - Sterile container (not to be placed in formalin) viii) If a prelimenary report is required please fill up two requisition forms.
3
ix) Gram stain report for samples sent in the morning in case of CSF, fluids, exudates, and any
other, if required- available in Bacteriology Lab. Room No. 316 / Room No. 320
B) REJECTION CRITERIA 1. Leaking containers/blood stained containers , Open mouth containers . 2. Missing / inadequate identification. 3. Inappropriate request Folley's cather tip, oral swabs etc.4. Incomplete forms (It is important to write relevant clinical details. It helps
in carrying out proper procedure and giving a useful report) Mention antibiotics given and if any special cases are required to be tested.
5. Insufficient quantity of sample and collected in inappropriate container 6. Unknown time delay . 7. Before rejection , mention the reason on the form and send it back to the right
place
COLLECTION AND TRANSPORT OF SAMPLES BLOOD 1. Should be drawn under aseptic conditions 2. Clean the skin with savlon, apply 2% Iodine, or 10% povidone, wait for a minute to dry and draw the blood. Clean the iodine with 70% alcohol or spirit. 3. Draw out 5 ml of blood for one blood culture bottle containing 50 ml of
BHI broth. For paediatric age group,1 ml in 10 ml of BHI broth in MaCartney's bottles may be added, if available. Otherwise the same inoculum may be added to 50 ml medium also.
4
4. For Bactec blood culture vial as per recommendations i.e 5-10 ml /1-3ml 5. Transport to laboratory , if not possible keep in incubator at 370C or keep at
Room temprature 6. NEVER REFRIGERATE. URINE 1. Mid-stream urine sample is collected after giving proper instructions to the patient. a) clean the genitalia properly.
b) collect a "clean-catch" mid-stream urine sample in a sterile container. Do not touch the inner side of the cap or bottle in case of cotton.
2. Transport immediately to the lab. if more than half an an hour delay is
expected, referigerate at 40C. It should be processed within 3-4 hours of collection.
3. Catheterised patients - Do not collect from collection bag or after opening
the closed drainage. Clean a area over the collecting tubes and puncture with the help of a sterile needle and syringe and draw out the sample.
4. Suprapubic aspiration under aseptic techniques may be done in infants and where mid stream results are doubtful. CSF/OTHER NORMALY STERILE BODY FLUIDS 1. Collect under aseptic condition and transported immediately to the laboratory
5
2. They should not be refrigerated if delay in transport is expected . Keep at room temperature/ in incubator at 370C PUS AND OTHER SWABS 1. Do not apply antiseptics before collection. Clean with Normal Saline. 2. In case of discharge, 1-2 ml of sample in a sterile container is preferable to swab. 3. If swabs are sent, 2 swabs should be sent one for direct examination and one
for culture. 4. Vaginal swabs should be high vaginal swabs preferably collected after actual visualisation. It should not touch the sides of the vaginal wall. 5.Throat swab (2 in numbers) should be collected under direct visualisation without touching the tongue or buccal mucosa. In cases of suspected diphtheria - Mention on the requistion form. SPUTUM
1. Coughed up specimen after rinsing the mouth may be sent in a sterile screw capped container.2.Adequate amount ( 2-3 ml), May be refrigerated up to 3-4 hours.
STOOL 1. Place a small quantity ( 1-2 gm) in a wide mouth clean container. If mucus or flakes present – must be included. 2.Not to be collected from bed pan, should not be contaminated with urine.
3.Must be transported within 1-2 hours of passage / Store at 2-80C
6
4. Rectal swabs - only if stool is not possible CATHETERS AND TIPS 1.Mark the junction of Skin and Catheter
2. Withdraw the catheter a little and cut it at about 1 cm distal to the mark .
3. Send 5 cm length of the catheter in sterile container. Transcutaneous part is a
better sample than the tip in case of long catheters. BACTERIOLOGICAL METHODS DIRECT EXAMINATION : 1. Gram's stain of Pus, other discharges, swabs, CSF and body fluids 2. Gram's Stain of sputum samples 3. Gram's stain from cultures 4. Wet preparations of urine 5. Hanging drop preparation 6. Albert's stain for C. diphtheria GRAM'S STAIN Preparation of smears for Gram staina) Swabs - If two swabs are provided ,one is cultured directly . The other is
used for making smear. Roll the swab lightly on the clean glass slide. If
7
only one swabs is taken, first it should be cultured and then used for smear preparation.
b) Pus and discharges – spread over a clean glass slide. The smear should not be
very thick. c) Sputum samples -Same as pus but if mucous is present it should taken as
a representative sample. c) CSF andClear fluids – Take a loopfull on a clean glass slide an let it air dry.
Air dry as such without spreading. Cultures - Take a loopful of broth culture and let it air dry OR take a loopful
of sterile normal saline on a slide. Touch the colony to be examined with the
loop and gently form a emulsion on the slide with the N. saline while spreading
it out (1x2 cm) STAINING PROCEDURE 1. Let the smear air dry. 2. Heat fix it by passing it over a flame 3-4 times, smear side upwards. The
heating should be enough for the slide to be tolerable to the back of the hand. 3. Flood with Crystal violet stain x 1min, wash in tap water 4. Flood with Gram's iodine x 1 min, wash in tap water.
8
5. Add Acetone over the smear and keep for a few seconds (10-15 sec) and wash off with water 6. Add safranin stain over the smear x 1min, wash. 7. Blot dry gently. Exam the smear under 10x and then add 1 drop of oil
( immersion oil / cedar wood oil) and examine under oil immersion lens. ALBERT'S STAIN
1. The smear is made similarly : smears are made on 2 slides - 1 for Gram's
stain and another for Albert's stain. Air dry and heat fix the smears. 2. Add Albert's I stain x 5-7 minutes and wash with tap water. 3. Add Albert's II stain x 1-2 minutes and wash with tap water 4. Gently blot dry it and examine. WET PREPARATION OF URINE
Take a drop of uncentrifuged urine on the clean glass slide and gently put a clean cover slip over it so that no air bubble is trapped in between. Examine under the 10x lens first and then 40x with the condenser lowermost .
One pus cell/ HPF and one bacteria / OIF is significant. HANGING DROP PREPARATION
It is used to examine motility of bacteria from broth cultures and
Direct stool exam of suspected cholera cases.9
1. Make a thin plasticine ring on the centre of a clean slide
2. Place a cover slip on a small piece of paper on the table. Place a loopful of
suspected stool sample in the middle of the cover slip. 3. Invert the slide with the plasticine ring over the cover slip preparation
and gently press to allow the coverslip to stick to the ring. 4. Gently turn the slide upside down so that the coverslip is upwards. 5. Examine under 10 x and focus the edge of the fluid. Then shift to 40x and
examine. PROCESSING OF SAMPLES BLOOD CULTURE Blood is sent in BHI broth / Bactec vial to the laboratory. On Receipt :
Kept in the incubator 370C.
After 24 hours - S/c done on Blood Agar and MacConkey Agar. If negative
After 48 hours - S/c done similarly.
05 days - Final S/c on BA and MA in case of positive cultures proceed 10
for identification and Antibiotic sensitivity test. Cultures observed -3 weeks in Infective Endocarditis patients where more
than one sample is received . Preliminary Report
Wherever a growth of suspected pathogen is detected, it should be
communicated. In case of suspected contaminated sample e.g. more
than 3 types of bacteria, diphtheroids, or Coag neg. Staphylococci, a repeat
sample should be requested.
If Salmonella is suspected slide agglutination done and reported. Final Report In case of positive cultures, the final report with identification and Antibiotic
sensitivity is dispatched. Additional Report The report after 10 days and 3 weeks is sent only if positive for suspected
pathogenic bacteria.
Automated Blood culture system( BACTEC 9120):-
11
Gram staining is done on getting positive signal followed by subculture on MacConkey agar and
Blood agar and SDA and identification and Antibiotic sensitivity testing is done
The Negative culture report is given after five days of incubation as per protocol of manufacturer.
Prolong incubation if fastidious organism is suspected. CEREBROSPINAL FLUID On receiving:- Centrifuge at 1500 rpm x 10 min. a) Deposit : Prepare smear for Gram staining and Culture on BA, CA , MA and BHI broth b) Supernatants : Shift in a sterile vial and can be used for Ag detection c) Add the rest to the deposit and incubate at 370C B. Keep the sample , inoculated Plates and Broth at 370C. CA is incubated in
candle jar. 1. Smear report- as Pus cells and Bacteria present ( Specify morphology and gram stain)
If smear suggestive of meningitis - direct sensitivity may be put up
Convey the preliminary report on telephone.
12
2. Observe cultures after 24 hours -
If positive - Identify, do the antibiotic sensitivity and send the report. If negative: Reincubate , look for any turbidity in broth or CSF and if
present get a subculture done.
Also if the direct smear positive and culture negative - get a subculture done from the sample.
3. If still Negative : Final report after 48 hours
3. In case of Coagulase negative staphylococcus, Diphtheriods or other
suspected contaminants ask for repeat sample 6. Diphtheroids should be differentiated from Listeria species Other Sterile Fluids Other sterile fluids are treated similar to CSF STOOL SAMPLE
Direct Hanging drop if V. cholerae is suspected. If positive,
immediately send a preliminary report Isolation 1. Direct inoculation on MA and DCA . BA and TCBS if V. cholera is suspected 2. BA inoculated when membranous colitis is suspected 3. Inoculate into Selenite `F' enrichment broth.
13
4. Alkaline peptone water if V. cholera is suspected ( re-examine after 3-4 hrs of incubation.) After 24 hours 1. Suspected Salmonella/Shigella colonies are identified and reported 2. Pure growth of any other Gram Neg. Bacteria in Children less than 3 years is
identified and reported. 3. Pure growth of staphylococci is reported. 4. Vibrio cholerae is reported. If present.
4. If not positive for Salmonella/Shigella- Do a subculture from Selenite `F'
enrichment broth. After 48 hours If none of the above is positive - Report as “No Salmonella/Shigella/ Vibrio
grown. A comment on normal flora can be made , if relevant. PUS, EXUDATES AND WOUND SWABS , CATHETERS/ DRAINAGE TUBES & TISSUES
If 2 swabs are received - one is cultured on Blood Agar (BA) and
MacConkey Agar (MA) by applying the swab on the primary inoculation 14
site on BA and MA and then streaked to get isolated colonies. The swab is also inoculated in TGB / RCM medium.
The other swab is used for preparing the direct smear.
If only 1 swab is received , it should be first cultured and then processed for smear.
B If pus discharge/aspirate/any other aspirate/ any other infected fluid is sent:- 1. One loopful is used to inoculate BA and MA and one loopful is put in TGB 2. One loopful is used for direct smear C If catheter or tube is sent :-
Roll the catheter all over with the help of a sterile forceps on the BA and then MA.
The catheter / tube is then put into the TGB medium.
15 colonies of bacteria growing on the plate are significant. D. The tissue should be minced in a sterile tissue grinder. Then process as :
1. One loopful is used to inoculate BA and MA and one loopful is put in TGB 2. One loopful is used for direct smear. Preliminary Report :
15
Direct Smear : If positive and suggestive of presence of bacteria a preliminary report may be dispatched. After 24 hours culture:
If growth occurs proceed for identification.
Identify and do Antibiotic sensitivity testing if 1 or 2 organism are grown.
3 or more types are reported as such and no complete identification and
antibiotic sensitivity reported. An indication of predominant bacteria
growing out of the 3 types may be given (e.g. predominantly GPC or GNB or Pseudomonas sp. ).
In case of diphtheroids - report as possible contaminants.
Coagulase negative staphylococcus from catheter may be significant.
Other sites may be reported as possible contaminants.
If the culture is sterile after 24 hours examine the Thio glycolate broth (TGB)
a) If Turbid - Subculture into BA and MA (may not give a true picture in
case of catheters where quantitative assessment is done) b) Meanwhile also reincubate the original plates
For Intravenous Catheter Tips 16
Do only QUANTITATIVE CULTURE BY ROLL PLATE METHOD.
a) If no blood culture submitted within 24 hours.
"Catheter tip culture in absence of concurrent positive blood culture has no clinical relevance. Kindly send a blood culture immediately".
b) If blood culture is negative'' Catheter tip culture in absence of concurrent positive blood culture has no clinical relevance".
c) If blood culture positive - report S/ST of blood culture only, however compare both.
SPUTUM / ENDOTRACHEAL SECRETIONS / BRONCHOALVEOLAR LAVAGE ETC, On receiving :
A. 1) Direct exam :
Make an uniform smear (from the purulent or blood stained part if present) on the slide. Examine the gram stained smear. In case of sputum ,first examine under low power objective 10X.
Study 5 separate fields. If epithelial cells are < 10 per field in 3 or more fields and Pus cells > 25 per field in 3 or more fields, then specimen is considered satisfactory. (This will be considered while reporting the culture results.
If GNB suggestive of Haemophilus species are there, a Chocolate agar
plate is inoculated and incubated in a candle jar.
17
B. 2) Culture
Inoculate sample into BA, MA and CA where indicated and incubate at 370C (in CO2 jar if CA is put) .
TGB to be inoculated in case of BAL, Endotracheal secretions or other
sterile samples.
B. At 24 hours, If culture positive - Report as discussed below : If culture negative i) Reincubate the plates ii) Subculture from TGB if turbid. At 48 hours- if culture positive 1. If pure growth or 2 types - Identify and do antibiotic sensitivity testing. 2. If scanty growth ( <20 colonies) correlate with smear and report the
identity with colony numbers. 3. 3 or 3 types = Report as Contaminants. If direct smear shows significant
number of Pus cells, then report as mixed flora with predominant colony type.
4. Pure diphtheroids/Coag Neg. Staph/ Viridans strep - Report as ? normal flora
18
5. In case of mixed normal flora growing, correlate with smear and ask to
submit a properly collected sample.
Rules for assessing the quality of Sputum Specimens
(i) Squamous Epithelial Cells - Reject if > 10SEC WBCs : Organisms : Sample :
(ii) Specimens contaminated with Epithelial cells represents oropharyngeal contamination, further processing would yield potentially misleading results.
Rules for Endotracheal Specimens
(i) Squamous Epithelial Cells - Reject if > 10SEC
Organisms - Nil /only yeast cells
(ii) Specimen contaminated with Epithelial cells unlikely to yield the significant results.
NASAL SWAB / THROAT SWAB Throat Swab
Direct smear : is only made if throat swab from suspected. Diphtheria case is sent. Albert's staining is done and send preliminary report .
19
Culture
Inoculate BA
If Diphtheria is suspected , Inoculate Loeffler's serum slope , 1/2 BA and 1/2 Potassium tellurite medium. Subculture from Loeffler's slope into the rest of 1/2 of Potassium tellurite medium and BA. Preferably within 4-6 hours of inoculation.
A. After 24 hours 1. Process B haemolytic strepto cocci, if grown. If isolation and grouping needs further 24 hours then a preliminary report should be dispatched.
2. In patients where chances of aspiration pneumonia are high, process pure cultures of Strep. viridans ,Gram neg. bacteria, or Streptococcus pneumoniae in immunocompromised patients. Also report of any alteration in normal flora.
3. If only normal flora:- Report as:
a) Non B haemolytic streptococci grown: Normal. No other common respiratory. pathogen grown.
b) If no growth is seen -Inappropriate sample . ? dry swab, Ask for repeat sample
Rules for assessing the appropriateness of Throat culture
Report the presence and absence of Group A Streptococci and Arcanobacterium hemolyticum only.
20
No Group A Streptococci and Arcanobacterium hemolyticum isolated on culture.
Nasal Swab
No smear is indicated
Culture on BA and MA Report: 1) If Staphylococcus aureus grows in culture- Report it. 2) Otherwise report as normal flora High vaginal Swab / Endo-Cervical Swab 1) Smear: may be made if indicated and on special request. 2) Culture : Inoculate on BA and MA. Inoculate Thioglycolate Broth also. 3) If after 24 hrs - sterile then S/c from TGB , If still sterile after 48 hours report as sterile. 4) Process : the following-
a) Pure GNB (1 types or 2 types)
b) B haemolytic group B streptococci 5) 3 or >3 types : Mixed flora should be reported as sample contaminated.
21
URINE 1. Direct Exam Place a loopful of urine ( Uncentrifuged ) on a clean slide, put a coverslip
and exam under 10 x and40x objective. Report :
1 pus cell/ HPF = significant
1 bacteria/ OIF = significant Direct sensitivity may be put up in such cases if only one type of bacteria is seen. 2. Culture
With 4 mm diameter loop of 26 gauge loop, inoculate 0.001 ml of
urine on to the CLED medium / MA. Complete transfer and immediate
streaking of the medium should be ensured. "Do not flame the loop in
between"
22
Reporting 1) 100 colonies - 105 bacteria / ml. consider this as significant. 2) 3 or more types of bacteria - Report as sample grossly contaminated. Ask
to repeat. 3) 3 or >3 types from post-op patients on long term antibiotics and/or
catheters - mention the morphological types of flora so as to give clinician some guidance in deciding antibiotics.
4) Process 1-2 types of bacteria and report antibiotic sensitivity. 5) Any growth on suprapubic aspriates and cystoscopic specimens is taken as significant 6) Patient on antibiotics and chronic pyelonephritis etc. or previous culture positive with same organism. low counts may be considered as significant and processed MEDIA :
The dehydrated culture media are prepared and sterilised as per manufacturer,s direction.
Blood Agar(BA)
Make nutrient agar. Autoclave it and then cool it at 450- 500C. Then in 100 ml nutrient agar add 5 ml
sheep blood. Blood should be taken from sheep in sterile conditions. Chocolate Agar (CA)
23
Heat blood agar at 800C for 20-30 minutes in water bath. Shake the agar in the interval of 10 minutes. Alkaline Peptone Water First make peptone water. Then adjust the pH 7.8.
Loffeler,s Serum slope: 1% Glucose broth - 25 mlSterile Serum - 75 mlMix both in sterile flask. Then inspissate at 800C for 45 minutes for 3 days.
STAINS : obtained commercially and prepared as per manufacturer directions. For Gram positive cocci: Tube coagulase –
Do not use contaminated plasma. First check the plasma with control strain.
First dilute plasma with normal saline - 1 ml plasma in 9 ml N/S. (1:10)
Take1 ml diluted plasma in sterile test tube. To this add 1-2 colonies of Staphylococci.
Incubate for 4 hour or over night at 370C. Then see the clot.
Do the test with controls also.
Catalase Test:-
24
Aseptically pick up small amount of bacterial growth with glass rod on the glass slide.
Add one drop of H2O2( 3%). Presence of air bubbling – positive test.
Keep the H2O2 in dark bottle and should be refrigerated when not in use.
Quality control check should be done daily with positive strain as H2O2 is unstable.
Catalase Negative : Perform the following tests:
Growth in 6.5% Nacl, Positive Bile esculin test, Positive Mannitol fermentation :-
- Enterococcus faecalis . Streptococcus pneumoniae (If colonies are alpha-haemolytic).:-
Do sensitivity with optochin disc and Put the plate in candle jar in (5% CO2).
Beta-haemolytic Streptococci-(If colonies are minute, beta haemolysis)
Do sensitivity with Bacitracin disc and Put the plate in candle jar
FOR SALMONELLA : S.Typhi - If no gas in glucose, H2S + , urea- negative, citrate- negative First do agglutination with poly `O' then O9 and then dH.
25
S. paratyphi A - If gas in glucose, urea, citrate negative. No H2S. Agglutination with Poly `O' then O2
and then aH. S. paratyphi B - If gas in glucose, urea negative. citrate + ve. Agglutination with Poly `O', O4, bH Growth On MacConkey agar LF Colonies - Take one single colony in peptone water for sugar and sensitivity tests. Sugar - mannitol motility agar, TSIA, Urea, Citrate NLF Colonies
Oxidase test is done.
LLF Colonies If oxidase test is negative, then test for sugar fermentation: Sugar - Full sugars - lactose, sucrose, glucose, mannitol motility agar, TSIA, PPA, urea, citrate
Oxidase Test positive - See the growth in 42 0C. No sugar fermentation:- Pseudomonas aeruginosa S. typhimurium –
Gas in glucose, urea negative, citrate positive and H2S in TSIA
26
Do agglutination with Poly `O', O4, iH antisera H. influenzae : Transparent colonies on CA. Gram Negative short bacilli, oxidase positive
Do sensitivity on CA. Culture on NA with X, V and XV disc. Test on BA with Staph for satellitism.
CAMP Test For CAMP test in the centre draw one line of staph aureus in the centre of BA
plate vertically and draw anoher line with group A horizontally .Do not touch
Staphylococcus aureus line. Draw a line with Grp B and then draw with test
organism. Do not touch the line with Staph. aureus. Put the plates in incubator at
37 0C. CAMP factor produced by Grp B Streptococci enhances the Beta lysin
produced by Staphylococcus and area of enhanced lysis ( arrow head) appear at the
junction of the two organisms
Antimicrobial Susceptibility testing: CLSI M100-S23 (2013) Performance
standards for AST :23 rd Informational Supplement guidelines Guidelines.
The accuracy and reproducibility of the test are dependent on maintaining a
standard set of procedures
that are standardized and followed in the laboratory.
The optimum media, inocula, antimicrobial agents, incubation and interpretation of
results are described .
27
Preparation of Plates
Muller-Hinton media is used. Defibrinated blood is necessary for testing fastidious
organisms such as Streptococcus, Enterococcus etc. in which cases Muller Hinton
plates supplemented with 5% sheep blood is used. pH of media is 7.2-7.4. The
medium is poured on to petridishes to a depth of 4mm .
In the smaller plates upto 6 antibiotic discs are applied whereas in the larger plates
upto 12 antibiotic discs
are applied. Poured plates are stored at 40C. Before inoculation plates are dried
with lid agar so that there
are no droplets of moisture.
Preparation of Inoculum
One or two morphologically similar colonies were picked up by the straight wire
and transferred to a test tube containing sterile normal saline. The density of the
suspension is standardized by further dilution if necessary to match the density
visually with 0.5 McFarland standard.
Inoculation
Plates are inoculated with a sterile cotton which is dipped into suspension and
surplus removed by rotation of the swab against the side of the tube above the fluid
level. The medium is inoculated by even streaking of the swab over the entire
surface of the plate in several directions.
28
Antibiotic discs
After the inoculum has dried, single discs are applied with forceps. Discs are stored
at 40C in sealed container and are allowed to come to room temperature before the
containers are opened.
Incubation - Plates are incubated for 16-18hours at 370C.
Reading of Zones of Inhibition
The diameters of zones are measured with a millimeter scale. The point of abrupt
diminution of growth, which in more cases corresponds with the point of complete
inhibition of growth is taken as the zone edge.
Interpretation
Each zone size is interpreted by reference table to one of the following categories
Susceptible - infection treatable with normal dosage.
Intermediate- Infection that may respond to therapy with higher dosage or if
infection is in a situation
where the agent is concentrated.
Resistant - not treatable with the agent.
29
Controls
Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Pseudomonas
aeruginosa ATCC27853 and Enterococcus faecalis ATCC29212 are tested daily
by the above mentioned procedure for the relevant drugs. Zone sizes are recorded
and if in three successive days the zone size for a particular drug/ drug falls outside
the susceptible range laid down by CLSI M100-S23(2013) Performance standards
for AST :23rd Informational Supplement guidelines for disc diffusion, the
technique is investigated for sources of error.
MEDIA Ability to support
1. MacConkey Agar E.coli, Staph.aureus, Salmonella typhi, E.faecalis
2. Blood Agar Streptococcus groupA
Blood Agar Streptococcus groupB
Streptococcus pneumoniae
3. Mueller Hinton Agar E.coli, Staph. aureus, Pseudomonas, E.faecalis
4. CLED E.coli, Staph.aureus, Salmonella typhi, E.faecalis
5. CA Haemophilus influenzae
ESBL Screening:-
30
Antibiotic disks
ceftazidime (CAZ) (30g), cefotaxime (30g) (Cefo) and Ceftriaxone (cet) (30g)
disks.
ceftazidime/ Clavulanic acid (30/10g) disks
.
Inoculum
The inoculum / incubation is according to the CLSI method of disk diffusion
testing.
Interpretation
1. A zone size of CAZ <=22mm, cefo<=27mm, cef <=25mm - Suspicious for
ESBL production.
2. A difference of >=5mm in zone diameter between CAZ disk and CAZ +
Clavulanic acid disk: –
- ESBL positive.(e.g. CAZ = 14mm, CAZ/Clav =21mm
OR
CAZ = no zone, CAZ/Clav = 6- 7mm: ESBL positive
Controls
Klebsiella pneumoniae ATCC 700603
31
(ESBL producer) is taken as control; zone size of K.pneumoniae ATCC 700603
CAZ - 10-18mm
Cefo - 17-25mm
Cet - 16-24mm
>=5mm increase in zone size with CAZ
>=3mm increase in cefo zone diameter.
Screening for MRSA:-
A) Disk diffusion susceptibility method
1)Oxacillin Disc diffusion test: 1 ug oxacillin disk is used on M-H agar. Incubate
for a FULL 24 hrs at 35° C.
Interpretation of results :
Resistant (MRSA): < 10 mm zone size of inhibition
Confirm with Oxacillin Screening Agar: 11-12 mm zone size of inhibition
Susceptible (No MRSA) > 13 mm zone size of inhibition
2 ) Cefoxitin (30ugm) Disc Method : Use Muellar Hinton agar plate and place
cefoxitin disc and incubate at 370 C for 24 hours.
Interpretation of results:
MRSA : Zone of inhibition < /= 21 mm
MR CONS: < /= 24 mm
B) Oxacillin Screening agar method:-
Preparation Mueller Hinton agar (MHA) with 4%NaCl.
- To 200ml distilled water; add 7.5gm MHA and 8gm NaCl.
-
32
Preparation of antibiotic stock.
- Prepare a stock solution of oxacillin at 1g/ml.
- Add 90l of this stock to 14.9ml of molten MHA (with 4%NaCl).
Shake well and pour onto petridishes. The final concentration of oxacillin
in the screen agar thus becomes 6g/ml.
Inoculum
Prepare the inoculum in normal saline (matched to a turbidity of 0.5 McFarland).
- Mark the screen agar plates into sectors.
- Apply one drop of the inoculum on to the marked sector.
- After overnight incubation of the plates at 37oC - Read the plates.
Interpretation
- A growth of bacteria on oxacillin screen agar indicates methicillin
resistance.or > 1 colony or light film of growth = oxacillin/methicillin-
resistant
Controls :- ATCC25923
Vancomycin Screen Agar
- Prepare a stock solution of vancomycin at 1g/ml.
- Add 90l of this stock to 14.9ml of molten Mueller Hinton agar.
Shake well and pour onto petri dishes - the final concentration of
vancomycin in the plate becomes 6g/ml.
Inoculum
33
- Prepare the inoculum in normal saline (matched to a turbidity of 0.5
McFarland)
- With a pipette - put one drop of inoculum on the vanco - screen agar
plate (the plate should be marked into sectors so that ~96 strains can be
tested on one plate)
- Incubate at 37oC overnight.
Interpretation
- A growth on vanco-screen agar indicates resistance to vancomycin.
Controls :- ATCC 29212
Disk Diffusion test for VRE:
Although this method has been shown to be unreliable for detecting resistance in strains with intermediate or low-level resistance to vancomycin, it remains an acceptable alternative for those laboratories not routinely performing MIC tests. A disk content of 30 μg should be used. Plates should be read using transmitted light rather than reflected light, since some vancomycin-resistant strains produce hazy growth with a larger zone of inhibition. In addition, disk diffusion plates should be incubated for a full 24 hours. MIC tests should be performed for strains demonstrating intermediate zones if vancomycin is being considered for treatment. Interpretation of Result: vancomycin resistance by disk diffusion (30 μg) ( 2004
NCCLS breakpoints)
34
Susceptible: ≥ 17 mm); Intermediate :15-16 mm ; Resistant :≥ 14 mm.
Procedure to follow when VRE are isolated from a clinical specimen:
Confirm vancomycin resistance by repeat testing. Assure culture purity by
performing a Gram stain of the growth from the vancomycin susceptibility
test. Common causes of false positive vancomycin resistance are mixed
culture (Gram negative rods or yeast mixed with enterococci) or
misidentification .
Hospital Infection Control Laboratory :
This section is involved with the surveillance of hospital infections in the Institute.
Environmental sampling is done to monitor effective decontamination and
disinfecting procedures carried out in the hospital. The sterility of the fluids
purchased is tested on the request of the Medical Supply Stores. Monitoring of
antimicrobial resistance is done by keeping a record of the resistance pattern of the
bacteria causing infections in our hospital. The outbreaks are identified and the
concerned unit informed in time to take timely control measures. Surveillance of
the staff to detect any carrier state in outbreaks is undertaken
Water Bacteriology: The bacteriological quality of water in the Institute hospital
and residential areas is undertaken to monitor quality of water being supplied to
the Institute.
35
EQUIPMENT TEMPERATURE MAINTAINING RECORD
Instrument…………… Selected
temperature………….
Room No…….. Observer………………
Date Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Date
1. 1.
2. 2.
3. 3.
4. 4.
5. 5.
6. 6.
7. 7. 36
8. 8.
9. 9.
10. 10.
11. 11.
12. 12.
13. 13.
14. 14.
15. 15.
16. 16.
17. 17.
18. 18.
19. 19.
20. 20.
21. 21.
22. 22.
23. 23.
24. 24.
25. 25.
26. 26.
27. 27.
28. 28.
29. 29. 30. 30. 31. 31.
37
38