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

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         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 .  

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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. 

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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.

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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 

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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

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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

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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.  

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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):-

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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. 

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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. 

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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 :

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 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.  

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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

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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 .

 

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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.

 

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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.                    

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 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" 

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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) 

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 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:-

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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.

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 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 

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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 .

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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.

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 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.

 

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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

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(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.

-           

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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

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-          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)

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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.

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 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.  

 

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