Tuesday, 30 August 2016

KNOW ABOUT DRIED BLOOD SPOT(DBS)



    DRIED BLOOD SPOT




a)difinition of DBS;-is a whole blood collected on a filter paper and dried which allows for easier transportation of the specimen.

b)Requirements during prepairing DBS
     1)Blood collection cards(filter paper)-is a paper like that are used in collecting blood.
    2)Sealable plastic bags-is a plastic bag used to store or collect all the requirement that are used in DBS.
    3)Glycine weighing paper-it stands between DBS cards for transporting/prevent coss contamination.
     4)Desiccant packs-when transporting from one place to another it helps to remove the excess moisture.
     5)Humidity cards-is a card on which a moisture sensitive chemical is impreginated that can change colour.also help to maintain moisture during transportation.
     6)Sharp containers-used for deposing the sharp instuments after work.
      7)Alcohol swab-used to clean the finger before pricking and after pricking.


C)Important tips for drying DBS.
   1)Avoiding touching or smearing the blood spots becouse it may lead to exploding of diseases in case the child has any diseases.
   2)it must dry completely before packagein order to be in safe order.
   3)heat is not allowed during drying process becouse it will deteriorate DBS.
    4)It must be away from direct sunlight becouse the light can give negative results.
    5)allow the specimen to fully air dry horizontally as it is adviced.

D)How to pack DBS storage after drying is complete.
     1)appropriately stock DBS
       -Glycine weighing paper must be placed on DBS to pevent cross contamination.
     2)insert into sealable plastic bags
        -Bags which are big in size must be avoided becouse cards will be shuffling inside.
     3)Desiccant packets shouold be added
        -Desiccant save the drying agent,some need more or fewer depending on environment and storage conditions.
     4)HUmidity cards.
       -humidity cards must be recharged before use and if a drying oven is not available,cards and excessivenumber of desiccant must be placed.


E)How to pack DBS for shipping.
  1-Insert bundled DBS into rip resistant envelope.
  2-appropriate documantation with shipment must be included.
  3-both into brown envelope and seal for shipping must be inserted

F)How to store DBS
1-it is acceptable to store DBS in a styrofoam box
2-avoid leaving in a motor vehicle,sun and heat will deteriorate DBS

3-spots in an mulfuctioning refrigerator where watermay drip on a soak the spots must be avoided.

Monday, 29 August 2016

UNDERSTAND ABOUT MICROBIOLOGY







MICROBIOLOGY









GENERAL PROCEDURES FOR CULTURING SAMPLES



Streak the right Plates with right Samples
 


After streaking incubate the plates at 37°C for overnight
 


 

      After incubation observe the plates for growth               

 

                                                                                                        
If there is no Growth, Report it as                                      If there is Growth, Do
NO GROWTH                                                                      confirmative tests and
                                                                          Sensitivity Tests


                                                                           
           DAY 3
 


Read the final identification, Sensitivity Plates
                                  And Give the Report


URINE

 

Day 1                                                                                              
REQUIREMENTS
CLED (Cystein Lysin Electrolyte Deficient media)                                                                                                                                                                  

METHOD
·         Give a number by registering in the culture register.
·Write the number on the sample and the plate used for                                                         ccultation.           
·         Heat the 5mm loop until red hot and allow to cool(do not shake)
·         Mix the sample, open the cap and take a loop full of urine and inoculate the half plate of the media.
·         Incubate at 37°C
·         Centrifuge about 5-10ml of the remaining urine sample and do the microscopy. Look for bacteria, pus cells and yeast cells.
·         Record the finding on the register and also the clinical SIGNIFICANT of the patient.

Day 2

Observation of Urine Culture in Cled Plates

  • Observe the CLED plates for growth.
  • Pick a single colony and do Gram stain
  • Observe the slide under microscope for cocci or bacilli
  • If Gram Positive cocci seen,check whether they are arranged in cluster or chain.
  • If seen in Cluster, it will be Staphylococcus species and if seen in chain, it will be Streptococcus species
  • For confirmation, carry out catalase test.
  • If catalase positive, it is Staphylococcus species and for confirmation of Staphylococcus aureus perform coagulase test.
  • If Catalase negative, it will be Streptococcus species.
  • If Gram negative bacilli seen in Gram stain smear, observe the plates for Lactose fermenting or Non Lactose Fermenting colonies.
  • In the plates, if Lactose fermenting colonies seen, observe for smooth yellow colonies and mucoid yellow colonies.
  • If smooth yellow colonies seen, it will be E.coli and for further confirmation carry out Bio – Chemical tests like KIA, MIU Citrate and Oxidase.
  • If mucoid yellow colonies seen, it will be Klebsiella species and for further confirmation carry out Bio – Chemical tests like KIA, MIU Citrate, and Oxidase.
  • If Non Lactose Fermenting colonies seen in the plates, do oxidase test.
  • If oxidase positive, it will be Pseudomonas species and if oxidase negative, it will be Coliforms.
  • Also carryout Antibiotic Sensitivity tests In Muller Hinton Agar Plates


Day 3

Read the final identification and also read the sensitivity plates and give the final report.



Possible Pathogenic organisms
Gram Positive                                                            Gram Negative
Streptococci                                                                 E.Coli
Staphylococci                                                             Klebsiella
Pseudomonas
Other coliforms






STOOL SAMPLES

Day 1


REQUIRMENTS
  • XLD
  •  Mac-Conkey
  • Selenite broth
  • TCBS
  • Alkaline peptone water.
METHOD
  • Give a number by registering on the culture register
  • Record the consistency  of the stool sample on the register
  • Heat the wire loop till red hot and allow to cool
  • Use full plates of XLD,TCBS and Mac-Conkey to inoculate
  • Inoculate ( about 1 gm of stool) into selenite broth and alkaline peptone water
  • Incubate the plates and the enrichment broths at 37°C for over night
  • The selenite broth and alkaline peptone water is sub cultured the next day on XLD and TCBS respectively.
  • Do the microscopy and rule out parasitic infection and look for pus cells

Day 2
Observation of Stool Culture in XLD Plates
  • Observe the XLD plates for lactose fermenting and Non – Lactose fermenting colonies.
  • If Lactose fermenting colonies seen, neglect the plate and sub culture the selenite enrichment broth on new XLD Plate and incubate.
  • Incubate the subcultured XLD plate for further day  and observe the plates for growth

  • If lactose fermenting colonies seen, give the report as No Salmonella, Shigella isolated       (NSSI )
  • If Non – Lactose fermenting colonies seen, observe for red, smooth colonies and red black centred colonies.
  • Also sub culture the selenite enrichment broth on new XLD Plate and incubate for further day.
  • If red, smooth colonies seen on XLD Plate, it will be Shigella species.
  • For confirmation carry out biochemical tests.
  • If red, black centred colonies seen, it will be Salmonella species.
  • For confirmation, do biochemical tests.
  • Also carryout Antibiotic Sensitivity tests In Muller Hinton Agar Plates for all organisms and for Streptococci do sensitivity in Blood Agar Plates.
Day 3
 

Read the final identification and also read the sensitivity plates and give the final report.

Possible Pathogenic organisms and Identification of Colonies
Gram Negative 
Shigella Sp                       
Salmonella Sp
Pathogenic E.Coli
Vibrio Sp

POINTS TO REMEMBER
*      NSSI -No  Salmonella, shigella isolated
*      For children less than 2 years, do stool culture in both XLD plates and Mac Conkey plates to detect E.coli.
*      If pure smooth, pink colonies seen without any other mixed growth on Mac conkey plates, it will be pathogenic E.coli.
*      Carry out biochemical tests in order to confirm pathogenic E. coli.
*      If mixed  growth is seen in Mac Conkey plates and Lactose  fermenting colonies  in XLD
       Plates report it as -- No Salmonella, Shigella and Pathogenic E.coli isolated.
*      For children below 2 years, rectal swab culture is also done in both XLD and Mac Conkey plates.



 BLOOD CULTURE
Day 1
Blood Culture Sample Collection   
For Pyogenic three samples, preferably three different sites or at least two different sites at intervals of one hour and label accurately  the name, age, date, diagnosis, site, lab no, time on all  blood culture sample.

MATERIALS REQUIRED
*      Spirit swab
*      Needle
*      Syringe
*      Blood culture bottle
*      Plaster

Keep all these things handy.

PROCEDURE
*      Wash hands with disinfectants
*      Palpate vein, keep the pricking point in mind.
*      Approximately clean 4-5cm circular area from centre to periphery.
*      Apply spirit swab
*      Wait for 5 minutes without touching any where.
*      Take out the cap from the needle and aspirate the blood according to the table.
*      Transfer the blood into the blood culture bottle and shake the bottle, opposite hand gently and horizontally.
*      The table shows volume of blood to be collected at different ages.



Age
Volume
2 years
1ml-1.5 ml
2-5 years
4ml
6-10 years
6ml
10 years
10ml

REQUIREMENTS
*      Oxoid Blood culture system media ( paediatric or adult volume)
*      Blood agar
*      Chocolate agar
*      Mac-Conkey agar

METHOD
*      1-10ml of blood is inoculated aseptically into the blood culture media according to the age of the patient
*      Register on the culture register and write the number on the bottle along with the date
*      Incubate the bottle at 37 °C for 7 days or until positive
*      The cultures are read every day for growth
*      When the indicator device shows growth and turbidity sub culture on blood agar, Chocolate agar and Mac-Conkey agar plates
*      Incubate the chocolate agar plate in the CO2 incubator
*      Incubate blood agar and Mac-Conkey agar plates at 37°C.

DAY 2

Observation of Blood Agar, Chocolate Agar, and Mac conkey Agar Plates
*      Observe the Blood agar, Chocolate agar and Mac conkey plates for growth.





Blood agar plate:
*      Observe the blood agar plate for golden, grey or white colonies.
*      Pick a single colony and do Gram stain.
*      If Gram positive cocci seen and arranged in cluster, it will be Staphylococcus species.
*      For confirmation,do Catalase test.
*      If Catalase positive, it will be Staphylococcus species.
*      Perform Coagulase test in order to confirm Staphylococcus aureus.
*      If Gram positive cocci seen and arranged in chain, it will be Streptococcus species.
*      For confirmation,do Catalase test.
*      If catalase negative, it is Streptococcus species.
Chocolate agar plate:
*      Observe the chocolate agar plate for small brown colonies.
*      Pick a single colony and do Gram stain.
*      If Gram negative cocco bacilli seen, it will be Haemophilus species.
*      For confirmation, carryout biochemical tests.
*      Final confirmation can be done by using X and V factor identification tablets or discs.
Mac Conkey agar plate:
*      Observe for Lactose fermenting colonies and Non – Lactose colonies.
*      If lactose fermenting colonies seen, observe for smooth pink colonies and mucoid pink colonies.
*      If smooth pink colonies seen, it will be E.coli.
*      For confirmation carry out bio chemical test.
*      If mucoid pink colonies seen, it will be Klebseilla.
*      For confirmation carry out bio chemical test.
*      If Non – Lactose fermenting colonies seen, it will be Coliforms or Pseudomonas species.
*      For confirmation, do Oxidase test.
*      If Oxidase positive, it is Pseudomonas species.
*      If Oxidase negative, it is Coliforms
*      Also carryout Antibiotic Sensitivity tests In Muller Hinton Agar Plates for all organisms, and for Streptococci do sensitivity in Blood Agar Plates.
*      For Haemophilus Sp do sensitivity in Chocolate Agar Plates
Note:
For identification of Salmonella species carry out bio-chemical test and agglutination test.
             The identification of Neisseria Sp can be done by using the particular anti sera agglutination test.
Day 3

Read the final identification and also read the sensitivity plates and give the final report.

Possible Pathogenic organisms and Identification of Colonies
Gram Positive                                       Gram Negative
Staphylococcus Sp                                     Salmonella Typhi (All Sp)                      
  Streptococcus Sp                                     Neisseria Meningitidis
      Enterococcus Sp                                        Haemophilus sp
                                                                  Pseudomonas  sp                                                                                                                                Klebsiela
                                                                  E.Coli
                                                     Other Coliforms                                 
Mycobacterium Tuberculosis and Fungal Sp.



CEREBROSPINAL FLUID


DAY 1
SPECIMEN COLLECTION
            CSF is obtained by lumbar puncture. These specimen are very precious and should be handled very carefully as these samples cannot be obtained easily. Usually 2 samples are sent to lab. The second sample is for microbiology. If only one sample is sent, the sample is first taken to Microbiology.

PROCESSING
*      Register in the miscellaneous register and give a number
*       Observe the colour, turbidity of the sample and record in the book
*      Take a sterile test tube and pour about 1ml of the CSF and centrifuge. If less sample is sent leave about 2-3 drops for cell count and centrifuge  the rest of the sample
*      Centrifuge at 1500rpm for 15 minutes
*      Decant the supernatant into another sterile test tube, and this can be used for the biochemical investigations
*      Mix the deposit with the remaining fluid, and use this for inoculation on 2 sets of full plates of blood agar, chocolate agar and Mac-Conkey agar. Prepare smear for gram’s stain from this deposit
*      Keep one set of the inoculated media in the 5 % CO2 incubator
*      Keep the other set of three plates in the routine incubator
*      Stain the slides for Gram’s stain and ZN stain ( if requested) and keep in the Microbiologist room

Note: Do not refrigerate CSF samples. Keep the sample in room temperature.


DAY 2

Observation of Blood Agar, Chocolate Agar, and Mac conkey Agar Plates
*      Observe the Blood agar, Chocolate agar and Mac Conkey plates for growth.
Blood agar plate:
*      Observe the blood agar plate for golden, grey or white colonies.
*      Pick a single colony and do Gram stain.
*      If Gram positive cocci seen and arranged in cluster, it will be Staphylococcus species.
*      For confirmation,do Catalase test.
*      If Catalase positive, it will be Staphylococcus species.
*      Perform Coagulase test in order to confirm Staphylococcus aureus.
*      If Gram positive cocci seen and arranged in chain, it will be Streptococcus species.
*      Do Catalase test for confirmation.
*      If catalase negative, it is Streptococcus species.
Chocolate agar plate:
*      Observe the chocolate agar plate for small brown colonies.
*      Pick a single colony and do Gram stain.
*      If Gram negative cocco bacilli, it will be Haemophillus species.
*      For confirmation, carryout biochemical tests.
*      Final confirmation can be done by using X and V factor identification tablets or discs.
Mac conkey agar plate:
*      Observe for Lactose fermenting colonies and Non – Lactose fermenting colonies.
*      If lactose fermenting colonies seen, observe for smooth pink colonies and mucoid pink colonies.
*      If smooth pink colonies seen, it will be E.coli
*      For confirmation carry out bio chemical test.
*      If mucoid pink colonies seen, it will be Klebsiella species.
*      For confirmation, carry out bio chemical test.
*      If Non – Lactose fermenting colonies seen, it will be Coliforms or Pseudomonas species.
*      For confirmation,do oxidase test.
*      If Oxidase positive, it is Pseudomonas species.
*      If Oxidase negative, it is Coliforms
*      Also carryout Antibiotic Sensitivity tests In Muller Hinton Agar Plates for all organisms and for Streptococci do sensitivity in Blood Agar Plates.
*      For Haemophilus Sp do sensitivity in Chocolate Agar Plates

Note: For identification of Salmonella species carry out bio-chemical test and agglutination test.
The identification of Neisseria Sp can be done by using the particular anti sera agglutination test.

Day 3
Read the final identification and also read the sensitivity plates and give the final report.
Possible Pathogenic organisms and Identification of Colonies
Gram Positive                                                    Gram Negative
Streptococcus Sp                                                 Nesseria Meningitidis
Staphylococcus Sp                                               Haemophillus Sp
                                                                            E.Coli
                                                                            Pseudomonas
                                                                            Proteus Sp
                                                                            Salmonella Sp
                                                                              Other coliforms

 

 

ALL KINDS OF TIP CULTURES AND SWAB CULTURES

 (PUS, WOUND SWAB, SKIN SWAB, NASAL SWAB)

DAY 1
REQUIREMENTS
*      Blood agar
*      Mac-Conkey agar
*      Heat sterilized forceps
PROCEDURE
*      Register in the miscellaneous register and give a number. Make sure to identify the correct type of the tip (e.g.; Foley’s Catheter tips, urinary catheter tip, ET tip, cup line etc.) from the request slip.
*      Heat the forceps until red hot and leave it until it cools.
*      Take out the tip using the sterile forceps and inoculate on full plate of blood agar, and Mac-conkey agar by rolling the tip on the surface of the plate media.
*      Incubate the plates at 37°C
*      Using a straight wire take out the Sample to inoculate and also prepare a Gram’s smear from this.
DAY 2
Identification of Organisms
Blood Agar plate:
*      Observe the blood agar plate and Mac conkey agar plate for growth.
*      In blood agar plate, observe for golden, grey or white colonies.
*      Now pick a single colony and do Gram stain.
*      If Gram positive cocci seen and arranged in cluster, it will be Staphylococcus species.
*      For confirmation, do catalase test.
*      If catalase positive, it is Staphylococcus species.




Mac-conkey agar plate:
*      Observe the Mac conkey plate for Lactose fermenting and Non – Lactose fermenting colonies.
*      If Lactose fermenting colonies seen, observe for smooth pink colonies and mucoid pink colonies.
*      If smooth pink colonies seen, it will be E.coli and for further confirmation carry out biochemical test.
*      If mucoid pink colonies seen, it will be Klebsiella, and for further confirmation carry out biochemical tests.
*      If Non – Lactose fermenting colonies seen, it will be coli forms or Pseudomonas species.
*      For confirmation,do Oxidase test.
*      If Oxidase positive, it is Pseudomonas species.
*      If Oxidase negative, it is Coliforms.
*      Also carryout Antibiotic Sensitivity tests In Muller Hinton Agar Plates for all organisms and for Streptococci do sensitivity in Blood Agar Plates.

Day 3

Read the final identification and also read the sensitivity plates and give the final report.

Possible Pathogenic organisms and Identification of Colonies

Gram Positive                                                Gram Negative
Staphylococcus Sp                                           Pseudomonas
Streptococcus Sp                                             E.Coli
                                                                        Klebsiella
                                                                        Other Coliforms



THROAT SWAB
DAY 1

REQUIRMENTS
*      Blood agar(full plate)

PROCEDURE
*      Register on the miscellaneous register and give a number
*      Write the clinical details of the patient on the register
*      Inoculate on a full plate of blood agar
*      Incubate at 37°C.
*      Prepare smear for Gram’s stain if it is requested

DAY 2

Observation of Throat Swab Culture in Blood Agar Plate

*      Observe the blood agar plate for alpha haemolytic, beta haemolytic and non – haemolytic colonies.
*      Pick up a single colony and do Gram stain
*      If Gram positive cocci seen and arranged in cluster, it will be Staphylococcus Species.
*      For confirmation do catalase test.
*      Perform Coagulase test in order to confirm Staphylococcus Aureus.
*      If Gram positive cocci arranged in chain, it will be Streptococcus Species.
*      For confirmation, do catalase test.
*      If catalase negative, it is Streptococcus Species.
*      For further identification of Streptococcal species, take two blood agar plates and one Bile Esculin plate and swab with culture. Add Optochin and Bacitracin discs on each Blood Agar plate and incubate the three plates (Two Blood Agar and one Bile Esculin) for over night.
*      After incubation, observe the blood agar plates for sensitivity with Optochin and Bacitracin discs.
*      If optochin disc shows sensitive, it is Streptococcus Pneumonia and if Bacitracin discs show sensitive, it is Streptococcus Pyogenes.
*      If all plates give negative result, do Streptococcus grouping test.
*      Also carryout Antibiotic Sensitivity tests in Muller Hinton Agar Plates for all organisms and for Streptococci do sensitivity in Blood Agar Plates.

Day 3

Read the final identification and also read the sensitivity plates and give the final report.
Possible Pathogenic organisms and Identification of Colonies
Streptococcus Sp
Candida Sp



OTHER SWAB CULTURES (Eye, Ear)
Day 1

REQUIREMENTS
  • Blood agar
  • Chocolate agar
  • Mac-Conkey agar

PROCEDURE
  • Register in the miscellaneous register
  • Write the clinical details in the register
  • Inoculate on blood agar and Mac-Conkey agar( half plates can be used)
  • Keep chocolate agar in CO2 incubator
  • Incubate BAP and Mac-Conkey at 37°C.
  • Prepare smear for Gram’s stain and keep on the plate. These smears will be stained if required. How ever requested Gram’s stains should be stained and kept in the microbiologist room.
DAY 2

Observation of Blood Agar, Chocolate Agar, and Mac Conkey Agar Plates

*      Observe the Blood agar, Chocolate agar and Mac Conkey plates for growth.

Blood agar plate:
*      Observe the blood agar plate for golden, grey or white colonies.
*      Pick a single colony and do Gram stain.
*      If Gram positive cocci seen and arranged in cluster, it will be Staphylococcus species.
*      For confirmation, do Catalase test.
*      If Catalase positive, it will be Staphylococcus species.
*      Perform Coagulase test in order to confirm Staphylococcus aureus.
*      If Gram positive cocci seen and arranged in chain, it will be Streptococcus species.
*      For confirmation, do Catalase test.
*      If catalase negative, it is Streptococcus species.

Chocolate agar plate:
*      Observe the chocolate agar plate for small brown colonies.
*      Pick a single colony and do Gram stain.
*      If Gram negative cocco bacilli, it will be Haemophilus species.
*      For confirmation, carryout biochemical tests.
*      Final confirmation can be done by using X and V factor identification tablets or discs.
Mac conkey agar plate:
*      Observe for Lactose fermenting colonies and Non – Lactose colonies.
*      If lactose fermenting colonies seen, observe for smooth pink colonies and mucoid pink colonies.
*      If smooth pink colonies seen, it will be E.coli.
*      For confirmation carry out bio chemical test.
*      If smooth pink colonies seen, it will be E.coli
*      For confirmation carry out bio chemical test.
*      If mucoid pink colonies seen, it will be Klebsiella species.
*      For confirmation, carry out bio chemical test.
*      If Non – Lactose fermenting colonies seen, it will be Coliforms or Pseudomonas species.
*      For confirmation,do Oxidase test.
*      If Oxidase positive, it is Pseudomonas species.
*      If Oxidase negative, it is Coliforms
*      Also carryout Antibiotic Sensitivity tests In Muller Hinton Agar Plates for all organisms and for Streptococci do sensitivity in Blood Agar Plates.



Day 3
 

Read the final identification and also read the sensitivity plates and give the final report.

Possible Pathogenic organisms (Eye, Ear)

EYE
                                                                          
Gram Positive                                          Gram Negative
Staphylococcus Sp                         Pseudomonas Sp
Streptococcus Sp                                       Proteus Sp
                                                                  Klebsiella Sp

EAR
Gram Positive                                          Gram Negative
Staphylococcus Sp                         Pseudomonas Sp
Streptococcus Sp                                       Proteus Sp
                                                                  Klebsiella Sp




DAY 1

REQUIREMENTS

*      Blood Agar
*      Chocolate Agar
*      Mac conkey Agar

PROCEDURE
*      Register in the miscellaneous register.
*      Write the clinical details in the register
*      Incubate the Blood agar, Mac Conkey agar and Chocolate agar plates with sample.
*      Incubate all the plates at 37°C
*      Prepare the smear for Gram’s stain and keep on the plate. These smears will be stained if required.
DAY 2

Observe the Blood Agar, Chocolate Agar and Mac Conkey Plates for Growth
Blood agar plate:
*      Take blood agar plate and observe for golden, grey or white colonies.
*      Pick a single colony and do Gram stain.
*      If Gram positive cocci, and seen in cluster, it will be Staphylococcus species.
*      For confirmation do Catalase test.
*      If catalase positive, it is Staphylococcus species.
*      Perform Coagulase test to confirm Staphylococcus aureus.
*      If Gram positive cocci and seen in chain, it will be Streptococcus species.
*      For confirmation do Catalase test.


Chocolate Agar Plate:
*      Take the Chocolate agar plate and observe for small, brown colonies.
*      Pick a single colony and do Gram stain.
*      If Gram negative cocco bacilli, it will be Haemophilius species.
*      For confirmation carry out bio chemical tests.
*      Final confirmation can be done by using X and V factor identification tablets and discs.
Mac conkey agar plate:
*      Take the Mac conkey plate and observe for lactose fermenting colonies and Non – Lactose fermenting colonies.
*      If lactose fermenting colonies seen, observe for smooth pink colonies and mucoid pink colonies.
*      If smooth pink colonies seen, it will be E.coli
*      For confirmation carry out bio chemical tests.
*      If mucoid pink colonies seen, it will be Klebsiella species.
*      For confirmation, carry out bio - chemical test.
*      If Non – Lactose fermenting colonies seen, it will be coli forms or Pseudomonas species.
*      For confirmation do Oxidase test.
*      If Oxidase positive, it is pseudomonas species.
*      If Oxidase negative, it is Coliforms.
*      Also carryout Antibiotic Sensitivity tests In Muller Hinton Agar Plates for all organisms and for Streptococci do sensitivity in Blood Agar Plates.
*    For Haemophilus Sp do sensitivity in Chocolate Agar Plates.




Day 3

Read the final identification and also read the sensitivity plates and give the final report.


Possible pathogenic Organisms
Gram Positive                                                            Gram Negative
Staphylococcus Sp                                                     Pseudomonas sp
Streptococcus Sp                                                       Klebsiella Sp
Enterococcus Sp                                                        Haemophillus Sp
                                                                                    Other Coliforms
                                                        


SEMEN CULTURE
DAY 1

REQUIREMENTS
  • Blood agar
  • Chocolate agar
  • Mac-conkey agar
PROCEDURE
  • Register in the miscellaneous register and give a number
  • Write the clinical diagnosis of the patient on the register
  • Inoculate on blood agar and mac-Conkey agar using a wire loop and incubate the plates at 37°C.
  • Inoculate on chocolate agar and keep the plate in CO2 incubator.
  • Prepare smear for Gram’s stain and keep on the plate. 

DAY 2
Observation of Blood Agar, Chocolate Agar, and Mac Conkey Agar Plates
*      Observe the Blood agar, Chocolate agar and Mac Conkey plates for growth.
Blood Agar Plate:
*      Observe the blood agar plate for golden, grey or white colonies.
*      Pick a single colony and do Gram stain.
*      If Gram positive cocci seen and arranged in cluster, it will be Staphylococcus species.
*      For confirmation, do Catalase test.
*      If Catalase positive, it will be Staphylococcus species.
*      Perform Coagulase test in order to confirm Staphylococcus aureus.
*      If Gram positive cocci seen and arranged in chain, it will be Streptococcus species.
*      For confirmation,do Catalase test.
*      If catalase negative, it is Streptococcus species.

Chocolate Agar Plate:
*      Observe the Chocolate agar plate for small Brown colonies.
*      Pick a single colony and do Gram stain.
*      If Gram negative cocco bacilli, it will be Haemophilus species.
*      For confirmation, carryout biochemical tests.
*      Final confirmation can be done by using X and V factor identification tablets or discs.
Mac conkey agar plate:
*      Observe for Lactose fermenting colonies and Non – Lactose fermenting colonies.
*      If lactose fermenting colonies seen, observe for smooth pink colonies and mucoid pink colonies.
*      If smooth pink colonies seen, it will be E.coli.
*      For confirmation carry out bio chemical test.
*      If mucoid pink colonies seen, it will be Klebsiella species.
*      For confirmation, carry out bio chemical test.
*      If Non – Lactose fermenting colonies seen, it will be Coliforms or Pseudomonas species.
*      For confirmation, do Oxidase test.
*      If Oxidase positive, it is Pseudomonas species.
*      If Oxidase negative, it is Coliforms
*      Also carryout Antibiotic Sensitivity tests In Muller Hinton Agar Plates for all organisms and for Streptococci, do sensitivity in Blood Agar Plates.
Day 3

Read the final identification and also read the sensitivity plates and give the final report.

Possible Pathogenic organisms and Identification of Colonies
Gram Positive                                                      Gram Negative
Neisseria Sp                                                                Pseudomonas
Streptococcus Sp                                                         E.Coli
Staphylococcus Sp                                                       Klebsiella
                                                                                    Other Coliforms
                                                                                    Haemophillus Sp

Fungal Sp

DAY 1

REQUIREMENTS
  • pH paper
  • Saline
  • Blood agar
  • Chocolate agar
  • Mac-Conkey agar
  • SDA
PROCEDURE
*      Register in the miscellaneous register and give a number.
*      Write the clinical diagnosis of the patient from the request slip to the register.
*      Inoculate on blood agar, chocolate agar, Mac-Conkey and SDA agar plates.
*      Inoculate the plate at 37°C, chocolate plate in CO2 incubator.
*      Make a smear for Gram’s staining and leave it in the hot plate.
*      Put the swab into about 500┬Ál-1ml saline, mix well.
*      Place a drop of saline onto a microscopic Slide, put a cover slip and observe under the Microscope.
*      Look for Trichomonas vaginalis, yeast cells, epithelial cells, clue cells and pus cells; record the findings on the register.
*      Touch the swab on a piece of pH paper and write the pH on the register.

DAY 2

Observation of Vaginal Swab Culture
*      Most probably vaginal swab culture shows commensals.
*      So only the possible pathogens are isolated and identified.
*      Neisseria gonorrhoeae can be identified by using antisera.
*      For other possible pathogens, carry out the procedure as that of common.
*      For all organisms,do the sensitivity on Muller Hinton Agar plates and only for Streptococcus.
*      Do sensitivity in Blood Agar plates.
Day 3

Read the final identification and also read the sensitivity plates and give the final report.

Possible Pathogenic organisms and Identification of Colonies
Neisseria Sp
Streptococcus Sp
Staphylococcus aureus
Candida Sp


Normal Flora in Vaginal Swab
Klebsiella,E.Coli ,Pseudomonas.(For these organisms give the report as Normal Vaginal Flora)



SAMPLE PICTURES


                                                                                                  
LF colonies in Mac conkey agar      Blood culture system showing Negative &                     Positive




Beta Hemolysis on Blood Agar                                                     Staphylococcus Species                                               
        
      

                                         Gram Negative Bacilli  




  • After putting for the antibiotic sensitivity, the inoculated peptone water broths used for ABST can be used for Biochemical reaction for the identification of the gram negative bacteria.
  • For lactose (LF) fermenting bacteria, citrate slant and motility butt is inoculated using the straight wire and Indole is tested the next day from the peptone water broth used for the ABST.
  • For non-lactose (NLF) fermenting colonies, citrate slant, motility butt, TSI slant and Urease butt is inoculated using a straight wire and Indole is tested from the peptone water broth the following morning.
  • The straight wire should be heated until red hot, cooled and then inoculated.
  • Before and after inoculating the mouth of the tubes should be flamed. This is to reduce the risk of contamination.
  • After stabbing the butt the slant should be streaked before taking out the wire.
Identification of certain Important Enteric Bacteria by Biochemical tests

S.No
Bacteria
Kligler  Iron Agar
Motility , Indole Urea Medium
Citrate
Oxidase
Slant
Butt
H2S
Gas
M
I
U
1
E. coli
A
A
-
+
+
d
-
-
-
2
Klebsiella
A
A
-
+
-
d
+
+
-
3
Entero bacter
A
A
-
+
+
-
-
+
-
4
Citro bacter
d
A
d
+
+
-
d
+
-
5
Salmonella
K
A
d
d
+
-
-
d
-
6
S. Typi
K
A
+
-
+
-
-
-
-
7
S. para typi
K
A
-
+
+
-
-
-
-
8
Shigella
K
A
-
-
-
d
+
d
-
9
Proteus
K
A
d
d
+
d
+
d
-
10
Vibrio
K
A
-
-
+
+
-
-
+

Symbols:       K - Alkaline ( red )  reaction , A - Acid ( yellow )


Reaction:      + (Positive reaction),- (Negative Reaction)


d = different biochemical  types



   Positive +
 

   Negative -
 

              
    TSI Showing H2S & Acid Productio

                                                                                 

        Reaction in citrate agar                                


Indole Positive and Negative 










MATERIALS USED:
·        MH media
·        Antibiotic discs( 6mm)
·        Peptone water broth
·        Sterile swab sticks
·        0.5 McFarland standard suspension

PROCEDURE
·        Inoculate 3-5 organisms to be tested into peptone water broth and mix by vortexing.
  • Incubate at 37°C for 2-4 hours.
  • Compare the turbidity with the standard suspension of 0.5macfarland. If too thin inoculate some more organisms and if too thick dilute with sterile peptone water.
  • Dip a sterile swab stick into the inoculated peptone water broth and remove the excess fluid by pressing the swab against the side of the tube above the level of the liquid.
  • Streak the swab all over the surface of the MH medium three times rotating the plate through angle of 60°C after each application.
  • Finally pass the swab around the edge of the agar medium.
  • Leave the inoculated plates to dry few minutes at RTP with lid closed.
  • Place the antibiotic discs using a sterile forceps or sterile needle tip. Disc dispersions also can be used. A maximum of 8 discs can be placed on the plate. Spaced evenly approximately 10mm from the edge of the plate, placing one disc at the centre.
  • Each disc should be pressed down gently to ensure even contact with the medium.
  • Incubate the plates at 37 °C (within 30minutes of inoculation) for overnight.






READING
  • After overnight incubation, measure the zone of inhibition in mm including the disc and record.
  • The measurement can be made with a ruler on the under surface of the plate without opening the lid.
  • Compare the reading with the standard chart of KB method.
  • Twice a month compare the sensitivity pattern techniques with ATCC strains.

REPORTING
Reporting is done in three ways.

  • Sensitivity or susceptible
An organism is called susceptible to a drug when the infection caused by it is likely to respond to treatment with this drug at a recommended dosage.

  • Moderately sensitive
                  When the susceptible drug is highly toxic this can be used.

  • Resistant
The organism is expected not to respond to a given drug, irrespective of the              dosage and the location of infection.

POINTS TO REMEMBER
  • Before inoculating and before reading, check whether the MH plates are contaminated.
  • Growth on the plate should be semi confluent.
  • Compare the sensitivity pattern and the techniques with the ATCC strains.
  • If all the primary antibiotics are resistant, higher antibiotic sensitivity test should be done.




Gram negative bacilli;
*      Ampicillin       Amoxicillin     Co-trimoxazole           Cephalexine    Norfloxacin                
*      Augmentin      Cefuroxime     Tetracycline                 Gentamycin     Cefotaxime
                                                                                   
For all urine samples include:
  • Ceftriaxone.
For Pseudomonas include: 
  • Ceftazidime
  • Diperacillin
  • Amikacin
For Gram positive cocci
·         Ampicillin      Amoxicillin     Methicillin       Oxacillin          Vancomycin               
·         Augmentin     Ciprofloxacin  Erythromycin  Gentamycin     Tetracycline
·         Cloxacillin      Cephalexine    Penicillin                                
                       
For Streptococcus include:
  • Bacitracin and Penicillin.

Higher antibiotics
  • Carbenicillin    Doxycycline    Chloramphenicol         Ceftazidime
  • Piperacillin      Ceftriaxone     Amikacin         Tobramycin


         
                 
                                                 Sensitivity in MHA Plate  
                     
                                                         Sensitivity in Blood Agar Plate

                                         
      Applying Antibiotic Discs in MHA               Sensitive and Resistant antibiotics    
                                      
 


PREPARATION OF MEDIA


MHA
Use: For antibiotic sensitivity testing.

Method
  • Read the instruction on the bottle carefully and follow them.
  • Suspend 3gm of dehydrated powder in 1L of distilled water.
  • Swirl to mix and leave for 10minutes on the bench.
  • Sterilize by autoclaving at 121°C for 15minutes.
  • Allow the media to cool down to 47°C.
  • Dispense to sterile Petri dishes under aseptic condition.
  • If there are bubbles remove them by flaming the surface briefly.
  • Record every thing in the media preparation register.
  • Take 2% of the prepared media for sterility testing.
  • Do the performance testing of the media using the ACTT strains (eg; Staphylococcus aureus and E.coli.)

BLOOD AGAR


Use: This is used to grow a wide range of pathogens particularly those that are more difficult to grow like H.influenza, Streptococcus pneumoniae, Neisseria species. This media is also required to detect and differentiate haemolytic bacteria especially, Streptococcus species.
Method
  • Read the instruction on the bottle carefully and follow them.
  • Weigh 37gm of dehydrated powder of blood agar base.
  • Add 1L of distilled water.
  • Leave for 10minutes to soak, swirl to mix.
  • Autoclave at 121°C for 15minutes.
  • Allow to cool to 47°C.
  • Add aseptically 70ml of 5-7% defibrinated blood.
  • Mix gently and dispense into sterile Petri dishes under aseptic condition.
  • Record every thing in the media preparation register.
  • Take 2% of the prepared media for sterility testing.

SELENITE BROTH

Use: A selective enrichment media for Salmonella species including S.typhi.

Method
  • Add 4g of Sodium Biselenite in 1L of distilled water.
  • Add 19gm of Selenite broth base and warm to dissolve.
  • Distribute to sterile tubes and sterilize for 10 minutes in a boiling water bath or free steaming.
  • Do not autoclave.

LOWENSTEIN JENSON MEDIA


Use: For the isolation and differentiation of Mycobacterium.

Method
  • Read the instruction on the bottle carefully and follow them.
  • Suspend 37.2g of LJ media powder in 600ml of distilled water with 12ml Glycerol.
  • Boil with constant agitation.
  • Sterilize by autoclaving.
  • Cool to 45-60°C, and aseptically add 1L of fresh egg suspension.
  • Mix well and dispense into sterile bottles.
  • Slant and coagulate at 85°C for 45 minutes.
  • Record every thing on media preparation register.


CHOCOLATE AGAR

Use: This medium supplies the factors required for the growth of Haemophilus
Influenzae. It also used to culture nutritionally demanding pathogens such
as Neisseria meningitidis and Streptococcus pneumoniae.

Method
  • Read the instruction on the bottle carefully and follow them
  • Weigh 37gm dehydrated powder of blood agar base
  • Add 1 litre of distilled water
  • Leave for 10 minutes to soak, swirl to mix
  • Autoclave at 121°C  for 15 minutes
  • Allow to cool until about 50°C
  • Add aseptically 70 ml of whole blood
  • Mix gently and heat in a 70 °C water bath until it changes to chocolate colour. This will take about 10 – 15 minutes. During that time the bottle should be mixed gently several times.
  • Allow the media to cool to 47°C , and dispense into sterile Petri dishes under aseptic condition
  • Record everything  in the  media preparation register
  • Take 2% of the prepared media for sterility testing
  • Do the performance testing of the media using the ATCC strains (Eg: H. influenzae, Neisseria sp, Strep. Pneumoniae )
  • Follow the sterility testing and the performance testing by recording the results on the register.

   Note: Alternatively excellent quality chocolate agar can also be prepared by
              incubating blood agar plates at 55°C for 30 minutes.




CLED


Use: A differential medium for the enumeration of urinary pathogens.

Method
·         Read the instructions on the media bottle very carefully
·         Suspend 36g of CLED media powder into 1L of distilled water
·         Leave on the bench for 10 minutes , swirl to mix
·         Autoclave at 121°C   for 15 minutes
·         Allow to cool to 47°C  , under aseptic condition , dispense into sterile Petri dishes
·         Record everything in the media preparation register
·         Take 2% of the prepared media  for  sterility testing
·         Do the performance testing of the media using the ATCC strains (eg Staph aureus  & E.coli )
·         Follow the sterility testing and the performance testing by recording the results on the Register.

SABAROUD DEXTROSE AGAR

Use: For the isolation and growth of fungus and yeast.

Method:
  • Read the instructions on the bottle carefully.
  • Weigh 65g of SDA powder into 1L of distilled water. Mix thoroughly, heat with frequent agitation.
  • Boil for 1 minute to completely dissolve the powder.
  • Autoclave at 121°C for 15minutes.
  • Record every thing in the media preparation register.

XLD

Use:  A selective media for the isolation of Shigella and Salmonella species.

Method:
·         Read the instructions on the bottle carefully
·         Suspend 53.5g of powder in 1L of distilled water.
·         Allow to soak for 10minutes.
·         Boil in water with frequent stirring.
·         Allow to cool to 47°C  , under aseptic condition, immediately  dispense into sterile Petri dishes
·         Do not Autoclave.

MAC-CONKEY AGAR


Use: For the isolation and differentiation of Enterobacteriaceae

Method
·         Read the instructions on the bottle carefully and follow them
·         Suspend 51.5g of media in 1L of distilled water
·         Leave for 10 minutes to soak, swirl to mix
·         Heat the water bath to dissolve
·         Autoclave at 121°C  for 15 minutes
·         Allow to cool to 47°C and aseptically pour into sterile Petri dishes
·         Record everything in the media preparation register
·         Take 2% of the prepared media for sterility testing
·         Do the performance testing of the media using the ATCC strains( eg: E. coli, Klebsiella, Pseudomonas, Proteus, Salmonella)
·         Follow the sterility testing and the performance testing by recording the results on the register.

          Note:   Media preparation register contains the following information:
                   Date/ Batch no / Total amount (L) prepared / Phys. Appearance /
                        pH / amount dispensed / Amount incubated / Sterility check for 24
                        Hrs / For 48 hrs / Performance testing strains results / Final
                        Comment
 



                     
    Media Preperation                                                                       Blood Culture system



  Agar Plates                                                                              Blood Agar


MOTILITY MEDIA


Use: To check the motility of bacteria.

Method
  • Dissolve 0.4 g and three pinches of Muller Hinton media powder in 100 ml of distilled water.
  • Boil to dissolve
  • Add into tubes.
  • Autoclave at 121°C to 15 minutes.

PEPTONE WATER


Use:  For carbohydrate studies and Indole test.

Method:
·         Dissolve 15 g of peptone water powder in 1 liter of distilled water.
·         Distribute in to test tubes.
·         Sterilize by autoclaving.


Use: For the differentiation of Enterobacteriaceae.

Method:
  • Suspend 24 g of citrate media in 1 litre of distilled water.
  • Heat to dissolve.
  • Dispense in to tubes.
  • Sterilize by autoclaving.
  • Allow the tubes to cool into slopes.
  •  

UREASE 
Use: For the detection of rapid urease production.
Method:
  • Weigh 2.1 g of urease agar powder into 95 ml of distilled water.
  • Soak and mix.
  • Sterilize by autoclaving.
  • Allow to cool and add 5 ml of sterile urease solution ( see below )
  • Dipense into sterile test tubes.
  • Urease solution:  Dissolve 20 g of pure urea in 50 ml of sterile distilled water Filter to sterilize.

TRIPLE SUGAR IRON (TSI):

Use: For the identification of Gram negative bacilli on the basis of dextrose, lactose and sucrose fermentation and H2S production.

Method:
  • Suspend 65 g of TSI powder into 1 litre of distilled water.
  • Mix and boil to dissolve
  • Distribute into test tubes.
  • Sterilize by autoclaving.
  • Allow the medium to set into slope with a butt about a 1 inch long.
General
·         All media should be kept inside the fridge and the media sterility forms should be filled every time a new batch is prepared.
·         Chocolates and Mueller Hinton media should be kept inside the incubator for overnight after prepared. The plates are checked the following day and those which show contamination are discarded and the remaining placed in the fridge

 

PREPARATION OF STAINS


1.                  LUGOL’S IODINE
           
Use: As a mordant in Grams staining

Composition:
                        10g                  potassium iodide
                        5g                    pure iodine
                        500g                distilled water
  • Grind iodine crystal and potassium iodide
  • Add a little water
  • Add the mixture into the remaining distilled water

2.         GRAMS DECOLOURISER

Use: For decolourising the smear after staining with crystal violet
           
Composition:
          2parts             Acetone
          1 part               Alcohol
  • Mix the two solutions




3.         CRYSTAL VIOLET
Use:  Stains gram positive bacilli
Compositions:
                        2.0g                 Crystal violet
                        20ml                Ethanol (95%)
                        0.8g                 Ammonium oxalate
                        80ml                Distilled water

  • Mix crystal violet with ethanol = solution A
  • Mix ammonium oxalate with distilled water = solution B
  • Mix solution A and B
  • Store the stain for 24 hours before use
  • Filter the staining in to the bottle for use

4.                  CARBOL FUCHSIN
           
Use: For staining of acid fast bacilli

Composition:
                        0.3g                 carbol fuchsin
                        10ml                ethanol/ methanol
                        95ml                distilled water
                        5ml                  liquid phenol
  • Mix carbol fuchsin with ethanol
  • Add distilled water and phenol filter before use






5.                  DILUTE CARBOL FUCHSIN

Use: Counter stain used in Grams staining

Composition:
                        1ml                  strong carbol fuchsin
                        9ml                  distilled water

  • Mix both the solutions


6.                  METHELENE BLUE

Use: Counter stain used in ZN staining
           
Composition:
                        0.3 g                Methelene blue
                        100ml              distilled water
  • Dissolve the methelene blue in distilled water
  • Filter into the staining bottle

7.                  3% ACID ALCOHOL

Use: Decolouriser used in ZN staining

Composition:
                        3ml                  HCL or H2SO4
                        97ml                ethanol / methanol
  • Mix the acid and the alcohol.


PREPARATION OF BUFFERED PEPTONE WATER

REQUIREMENTS
  • Peptone                       10.0g
  • Sodium chloride          5.0g
  • Phosphate buffer         10.5g

DIRECTIONS
  • Suspend 25.5g in 1L of purified water. Mix thoroughly.
  • Heat with frequent agitation and boil for 1minute to completely dissolve.
  • Adjust pH to 7.2+/-0.2 at 25°C.
  • Dispense 7ml into each 15x125mm screw capped tubes and loose caps.
  • Autoclave at 121°C for 15minutes. Do not over heat.
  • Let cool and tighten the caps.
  • Store at 2-6°C.

PREPARATION OF ALKALINE PEPTONE WATER (0.5% APW)
For enrichment in the isolation of vibrio species.

REQUIREMENTS
  • Peptone                       10.0g
  • Sodium chloride          5.0g
DIRECTIONS
  • Suspend all ingredients in 1L of purified water. Mix thoroughly.
  • Heat with frequent agitation and boil for 1minute to completely dissolve.
  • Adjust pH to 8.4 at 25°C by adding many drops of NaOH.
  • Dispense 5ml into each 15x125mm screw cap tubes, and loose caps.
  • Autoclave at 121 °C for 15 minutes. Do not over heat.
  • Let cool and tighten the caps.

STAINING TECHINIQUES

Gram Staing Technique

PRINCIPLE
Differences in Gram reaction between bacteria is thought to be due to differences in permeability of the cell wall of Gram positive and Gram negative organisms during staining process

REAGENTS USED
  • Crystal violet
  • Lugol’s iodine
  • Acetone- alcohol
  • Diluted Carbol Fuchsin

PROCEDURE
  • Make a bacterial smear and allow it to air dry
  • Heat fix the smear or fix with Methanol
  • Cover the smear with crystal violet for 30seconds
  • Wash off the stain with tap water
  • Tip off the water remaining on the slide and cover the smear with Lugol’s iodine for 30 seconds
  • Wash off with clean tap water
  • Decolourise rapidly with Acetone –Alcohol for 12seconds and wash with tap water
  • Tip off the excess water and cover the smear with diluted Carbol fuchsin for 30 seconds
  • Wash with running tap water
  • Wipe off the back of the slide and place it in a draining rack for it to get air dried
  • Examine under the microscope first with the high power to check the staining and to see the distribution of the material and then under the oil immersion to report the bacteria and cells.


RESULT
Gram positive bacteria                 Dark purple
Yeast cells                                     Dark purple
Gram negative bacteria                Pale to dark red
Nuclei of pus cells                        Red
Epithelial cells                              Pale red

REPORTING
The reporting should include:
Number of bacteria present                   many/ moderate/ few or scanty
Gram reaction of bacteria                      Gram positive/ Gram negative
Morphology of the bacteria                    Cocci/ Diplococci/ Streptococci/Rods or                                                                                    Coccobacilli.
Presence of Yeast cells and Epithelial cells

POINTS TO REMEMBER
·         Always check new batches of stain and reagent using smear with known gram positive and gram negative
·         If the smear is too big the gram negative organism may not be fully decolourised and may appear as gram positive
·         Gram positive organisms may appear as gram negative organism due to the following reasons
1.      cell wall is damaged due to antibiotic therapy or excessive heat fixing
2.      over decolourisation of the smear
3.      iodine solution used is too old
4.      smear has been prepared from a very old culture





Ziehl-Neelsen Staining Technique
                                   
This technique can be used to stain Mycobacterium tuberculosis (AFB)

REAGENT USED
  • Strong Carbol fuchsin
  • 3% Acid Alcohol
  • Methelyne blue

PREPARATION OF THE SMEAR
  • Select a new unscratched slide and label the slide with the serial number
  • Make the smear from the yellow purulent portion of the sputum using the wooden stick( tongue depressor) provided for it
  • A good smear is spread evenly, 2cms x 3cms in size and is neither too thick nor too thin. The optimum thickness of the smear can be assessed by placing the smear on a printed matter, the print should be readable through the smear
  • Let the smear air dry for a few minutes
  • Keep the slide on the hot plate for complete drying and fixing
  • Alternatively the smear can be fixed in Methanol

STAINING PROCEDURE
  • Keep the fixed slides on the staining rack
  • Flood the smear with strong Carbol fuchsin
  • Heat the slide underneath until vapour starts rising. Do not let the Carbol fuchsin boil. Leave for 10minutes
  • Wash in running tap water to remove the stain. Tip off the excess water. The slide will look red at this point.
  • Decolourise with 3% Acetone-Alcohol rinsing in running tap water at intervals over a period of 2-4 minutes(this step is very important)
  • Wash thoroughly in running tap water. Tip off the excess water from the slide
  • Counter stain with 1% Methylene blue for 2-3 minutes
  • Wash with running tap water and tip off the excess water
  • Wipe the back of the slide and keep on a tray for drying
  • After drying examine the slide under the microscope using 40xlens to select the suitable area of the slide and examine under the 100x lens using a drop of immersion oil.

RESULT
Acid –fast bacteria                                                    red (straight or slightly curved rods                                                                                                occurring singly or in groups)
Non acid-fast bacteria and background                 blue

REPORTING
If any definite bacilli are present, report the smear as AFB positive and give an indication of the number of bacteria present
  • 0-10 AFB/field                                   +++
  • 1-10 AFB /field                                  ++
  • 10-100 AFB/ 100field                                    +
  • 1-9AFB/100 field                               report  the exact number

POINTS TO REMEMBER
·         If only one or two bacilli are present request a further specimen
·         Tap water sometimes contain AFB that resembles tubercle bacilli
·         Occasionally stained scratches on a slide can be mistaken for AFB although these tend to be in a different focal plane to the smear
·         If no AFB is seen after examining the smear, report the smear as ‘No AFB seen’. Do not report as ‘negative’ because organism may be present but not seen in those fields examined.
·         Always check newly prepared stains with known high and low AFB positive sputum specimen
·         The heating step of the staining procedure should be carried out very carefully
·         The preparation of the smear should be carried out inside the safety cabinet.

HANGING DROP METHOD

PURPOSE
To see whether an organism is motile or non- motile. It is done to see the motility of the Vibrio Cholerae from stool sample suspected for Cholerae.

PROCEDURE
  • Transfer a loop full of fluid culture or water of condensation or fluid part of stool sample on to clean cover slip.
  • Place melted Vaseline on the 4 corners of the cover slip.
  • Place a slide (preferably a cavity slide over the cover slip and press.)
  • Turn over the slide so that the cover slip is upper most and see that the drop of fluid do not touch the slide and keep the slide on the microscope stage.
  • With the light reduced by closing the iris diaphragm, bring the edge of drop into focus with the low power(x10) objective.
  • Carefully swing the high power (x40) objective into position and again bring the edge of the drop into focus. Adjust the iris diaphragm to give the best illumination. Focus by using the fine adjustment of the microscope. Move the field of view to the centre of the drop since the organism near the edge of the drop may be non-motile, due to drying out or surface tension.

RESULT
  • Motile organisms are those which definitely move in relation to other organism from one point of the field to another.
  • One motile organism seen in a loop full of pure culture is sufficient to indicate that the culture is that of a motile organism.
  • Non-motile organisms may exhibit Brownian movement (bombardment of water molecules) they do not change the position in relation to the other organism in the field of view.

OXIDASE TEST

PRINCIPLE
            This is used as a differentiation test mainly for the aerobic and facultatively anerobic groups of gram negative bacteria.
            If a bacterium produces oxidase enzyme the reagent TPD will form a purple colour when it comes in contact with the oxidase producing organism.
Coliforms are oxidase negative while Pseudomonas is oxidase positive.

REAGENT
  • 1% TPD reagent (freshly prepared)

PROCEDURE
Filter paper method
  • Place a drop of 1% TPD reagent on to a piece of filter paper on a glass slide.
  • Using a glass rod or wooden stick take the suspected colony and apply on the wet area of the filter paper and observed for development of colour.

RESULT
POSITIVE REACTION                   Indicated by development of purple colour within                                                                        10 seconds.
NEGATIVE REACTION                 NO development of colour.

POINTS TO REMEMBER
  • Reagent can be directly put on to the suspected colony and observed for colour development.
  • Colonies taken from blood agar plate are unsatisfactory. Use of chocolate agar or Mac-conkey agar is the alternative.
  • For picking up colonies, iron containing wires are not recommended. Glass rods can be used.

CATALASE TEST

RPINCIPLE
Catalase is an enzyme present in some bacteria. Hydrogen peroxide produces an oxidative end product of the aerobic break down of sugars. If allowed to accumulate, this becomes toxic.
This test is mainly done to differentiate Staphylococcus (catalase positive) and Streptococcus (catalase negative).
REAGENT
Hydrogen peroxide
METHOD
  • Place a drop of Hydrogen peroxide on a glass slide.
  • Transfer the suspected colony from the culture plate on to the drop of Hydrogen peroxide using a glass rod or wooden stick and observe for immediate bubbling.
  • Alternatively place a drop of Hydrogen peroxide directly on to the suspected colony on the plate and observe for immediate bubbling.

RESULT
POSITIVE REACTION-                              Immediate gas bubbles                    
NEGATIVE REACTION-                            No gas bubbles or delayed gas production.

POINTS TO REMEMBER
  • Iron containing wires should not be used to pick colonies. Glass rod or wooden stick can use.
  • Colonies from blood agar plate are unsatisfactory for this test. They may yield false positive results, because the medium itself contains sufficient catalase. This can be tested by pouring Hydrogen peroxide on to the medium itself.

CONTROLS
Staphylococcus                       Positive for catalase


COAGULASE TEST

PRINCIPLE
  • To detect the ability of an organism to clot plasma by the action of enzyme coagulase.
·         The tube test shows the activity of free and bound coagulase.
·         Slide test detects bound coagulase.
·         Negative slide test should be check by tube test for free coagulase.
·         Controls should always be included.
·         This test is used to differentiate Staphylococcus aureus from other Staphylococcus.

MATERIALS REQUIRED
  • Fresh plasma from whole blood
  • Saline solution
  • Test culture

PROCEDURE (slide test)
  • Place two loopful of saline onto a slide and prepare a heavy, almost milky homogenous suspension of the bacteria to be tested.
  • Place two or three loop full of oxalated or citrated plasma to make a drop next to the test suspension.
  • Carefully mix the plasma into the suspension of organisms.

RESULTS
Visible clumps appearing within 5-20 seconds                   POSITIVE
No clumps                                                                              NEGATIVE

TUBE COAGULASE TEST FOR SLIDE NEGATIVES

PROCEDURE
·         Dilute plasma 1:10 with normal saline.
·         Take two test tubes marked T and C.
·         Add 0.5ml of diluted plasma to the two tubes.
·         Add 5 drops of test culture to the tube marked T only.
·         Mix gently and incubate at 37°C for 2 hours.

RESULT
POSITIVE REACTION-                  Shows complete clotting
NEGATIVE REACTION-                shows no clot

POINTS TO REMEMBER
  • Slide negative strains may become tube positive.
  • When plasma is diluted false agglutination do not occur.
  • All slide negatives should be reconfirmed by the tube test.

SKIN SCRAPING FOR FUNGUS

FOR SKIN
Sample collection:
  • Clean the area with sterile saline.
  • Inspect the lesion; go from the normal skin to the inflammatory junction.
  • Scrap the epidermis.
  • Do not go for dermis( bleeding should not occur at lesion)

PROCEDURES
  • Put one drop of 10% KOH on a clean glass.

  • Transfer the material on slide.
  • Keep a cover a glass.
  • Allow to dissolve all the keratinised material.
  • If culture is requested, inoculate into SDA media and incubate for 7 days at 37°C.


READING
  • Examine under the microscope for fungal hyphae and spores, under high power objective.

FOR NAIL
  • If surrounded skin is not inflamed, use spirit for cleaning the nail.
  • Allow spirit to evaporate. Take clipping with a nail cutter if nail is affected proximally( at end)
  • If lesion is present at nail bed use scalpel blade for scraping.
  • Use 20% KOH; allow dissolving the nail for 30 minutes.
  • Transfer a drop of the dissolved nail on to a slide. Put a cover slip and observe under the microscope for hyphae and spores.


GERM TUBE TEST

PRINCIPLE
Candida albicans produce a pseudogerm tube when inoculated to serum

MATERIALS REQUIRED
  • Human serum
  • Tubes
  • Slides and cover glass


PROCEDURE
  • 0.5ml of human serum is placed into a test tube.
  • Few colonies of yeast are suspended into it.
  • The tube is then placed in a water bath and incubate for 2 hours.
  • After 2 hours a drop of the suspension is placed on a glass slide, a cover slip is placed over it and examined under the microscope for the formation of germ tubes.

POINT TO REMEMBER
  • Tubes incubated should not be delayed in examining, as germ tube is a filamentous extension of the yeast cell and mycelium formation can obscure the reaction.


MENINGOCOCCAL ANTIGEN TEST

SPECIMEN COLLECTION
  • Body fluid samples(CSF, Serum, Urine)
Testing should be carried out as soon as the samples is collected other wise the     samples can be stored at 2 to 8°C for over night.
  • Blood culture samples should be incubated at 37°C for 24 hours before testing.

REAGENTS
  • Test latex
  • Control latex
  • Polyvalent positive control
  • Negative control

PROCESSING OF THE SAMPLE
Body fluids must be heated before testing.
  • CSF and Urine- heat the sample for 5minutes in a boiling water bath. Cool the sample to room temperature and clarify by centrifugation or membrane filtration.
  • For serum- add 3 volumes of 0.1 MEDIA to 1 volume of serum. Heat the sample for 5minutes in boiling water and cool to room temperature.
  • Blood culture samples. Centrifuge a 1-2ml sample to pallet the red blood cell for example at 1000g for 5-10minutes. Perform the latex test on the supernatant. If a non-specific reaction occurs heat the sample in a boiling water bath for 5 minutes and cool to room temperature and centrifuge and perform the test.
PROCEDURE FOR THE TEST SAMPLE
  • Process the sample as described above
  • Shake the latex reagents
  • Place one drop of test latex in one Circle and one drop of control latex on another Circle.
  • Using a disposable dropper, dispense 1 drop (40ul approximately) of test sample next to each drop of latex.
  • Mix the contents of each circle with a mixing stick and spread to cover the complete area of the circle use separate sticks for each circle.
  • Rock the card slowly and observe for agglutination for 3 minutes, holding the card at normal reading distance (25-35cm) from the eyes.
  • Discard the used reaction card for safe disposal.

PROCEDURE FOR POSITIVE CONTROL

The reaction of the test latex can be confirmed by adding polyvalent positive control to a reaction circle in which the test sample has not agglutinated the test latex after 3 minutes.
·         Use a disposable dropper bottle to add 1 drop of positive control to the circle containing test latex and specimen.
·         Mix using a mixing stick and discard for safe disposal.
·         Rock the card manually or place on a rotator for further 3 minutes. After this time definite agglutination should be visible in the test latex.
·         Discard the used reaction card for safe disposal.

PROCEDURE FOR NEGATIVE CONTROL
  • Place 1 drop of test latex in one circle on reaction card.
  • Dispense 1 drop of Negative control or uninoculated blood culture medium next to the test latex.
  • Mix using a mixing stick and discard it for safe disposal.
  • Rock the card manually or with rotator for 3minutes.
  • After this time there should be no significant agglutination in the test latex.

READING OF TEST RESULT
            A positive result is indicated by the development of an agglutination pattern within 3 minutes if mixing the latex with the test sample, showing clearly visible clumping of the latex particles.
The speed of appearance and the quality of agglutination depend on the strength of the antigen. Varying from large clumps which appear within a few seconds of mixing to small clumps which develop rather slowly
MANTOUX TEST
Procedure
  • Clean the dorsum of the forearm with spirit.
  •  0.1ml of (10TU or 5 TU) PPD should inject intradermally
  • Give instructions to the patient to keep the area protected from any contact till the reading is taken.

READING
  • Within 48-72 hours measure the zone of indurations.
  • For immunocompetent patient 10mm or more than 10mm is significant.
  • For immuno compromised patients 5mm is significant.

REPORT
  • After 48 hrs check the injected place and measure the Induration
  • If positive there will be swelling redness and itchiness on the concerned place
  • Measure the Zone of Induration in Millimetre by using a Ruler
  • Induration more than 10 mm is considered as Positive and Less than 10 mm is considered as Negative.



 FUMIGATION
Every week or every other week Thursday, Microbiology room must be fumigated.

CHEMICALS USED FOR FUMIGATION
  • Potassium permanganate                     75gms
  • Formalin(concentrated )                      140ml
PROCEDURE
  • Wipe the benches and other surfaces areas with 1% Sodium Hypochlorite.
  • Take all the register to the 127 area .Shift the gas cylinder and burner, sample trays, loops, lighter etc and any other thing that may be needed while the fumigation is going on to the place identified(previous grossing area)
  • Weigh 75gms of potassium permanganate and put in a tray (aluminium tray will be suitable)
  • Measure 140ml of Formalin in a jar and keep ready inside the Microbiology lab.
  • Keep the plaster ready to seal the slides of the door (better to cut the plaster and keep)
  • Seal  any place that fumes may come out ( eg: the opening at the bottom of the door )
  • Keep the aluminium tray with the Potassium permanganate on the floor in the middle of the room.
  • Switch off the lights and the AC and make sure that the safety cabinet, hotplate and the microscope is switched off.
  • Keep a label ready with the fumigated date and time on it, to paste on the door.
  • Pour the Formalin as quickly as possible and come out of the room.
  • Seal the slides of the door with the plaster whish was cut and kept. Paste the label on the door.
  • Fumigation is kept until Saturday morning. Early on Saturday morning the door is opened and kept until the remaining fumes go away and can work inside the lab comfortably.