UNDERSTAND ABOUT MICROBIOLOGY
MICROBIOLOGY
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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
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 500µl-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
|
||||||||||||
|
|
Reaction in citrate agar
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.
- 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
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.
- 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.
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.
- 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.
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