TCBS VIBRIO CHOLERAE SCREENING

Procedures:
Inoculate sample from patient onto TCBS agar and spot for yellow colonies
Suspicious Vibrio cholera will be sub on blood agar and nutrient agar
Perform V. cholerae serotyping.
Oxidase test will be carried on blood agar. If positive oxidase, perform full
identification and antibacterial susceptibility test.
CONCLUSION:
Sample from patient received.
General procedure, Gram stain:
Gram positive:
Catalase test
In cluster (gram positive coccus)
Catalase positive (Staphyllococcus group)
Run coagulase test, latex and rapid plasma, if all positive
Staphylococcus aureus, if negative, considered as CONS
(Coagulase negative Staphylococcus)
o Catalase negative (streptococcus group)
Inchain (streptococcus; divided into 3 Beta, alpha and
Gamma(
o Beta group (lancefield group):

Group A - Streptococcus pyogenes

Group B - Streptococcus agalactiae

Group C - Streptococcus
equisimilis, Streptococcus equi, Streptococcus
zooepidemicus, Streptococcus dysgalactiae

Group D - Enterococci, Streptococcus bovis

Group E - Streptococcus milleri and mutans

Group F - Streptococcus anginosus

Group G - Streptococcus
canis and Streptococcus dysgalactiae

Group H - Streptococcus sanguinis

Group L - Streptococcus dysgalactiae

Group M - Streptococcus fryi sp. nov

Group N - Lactococcus lactis

o
o

Group R&S - Streptococcus suis

other Streptococcus species are classified as

'non-Lancefield Streptococci
Gamma: PYR positive - Enterococcus
Alpha: optochin test;
Streptococcus pneumonia: sensitive to optochin
test
Streptococcus viridans: resistant to optochine

Gram negative bacteria:

Oxidase test (non lactose fermenter and proceed with biochemical test
(TSI, Urea, MR, Citrate, SIM)
STOCK CULTURE MEDIA
Introduction:
Sotck culture lab is a lab running all the quality check for media prepared by the
media preparation lab and maintaining stock culture of variety species of
bacteria by subculture periodically. Usually the stock that was kept will be use as
future reference or studies.
Techniques:
Inoculation: full plate streaking should sterilize loop after 2 quadrants
streaking while half plate such as BaMac advisable to sterilize by each
quadrant.
Aim: to ensure single pure colony successfully isolated.
Subculture of Neisseria gonorrhoea: this inoculation will be done one
duplicate and different type of media since it is a very sensitive bacteria
species. Subculture will be done on TMA and BA/CBA.
Prcoess:
Quality control: Quality control is done to check the quality of the media
prepared by lab preparation media in which the media will be inoculated
with the strain of bacteria that only able to live on the specific media. This
is to test the quantity of the nutrient inside the media is sufficient to
support the growth of desire microorgranisms.
Usually QC test will be run after 24hours of sterility test to make sure there
are no possible contamination.
Quality control for in house media:
Media
Test organism
Blood agar

Chocolate
blood agar

Expected reaction

Streptococcus
pneumoniae

Small, clear colonies

exhibit beat
haemolysis.

Streptococcus group A

Small to medium
colonies exhibit alpha
haemolysis
Growth. Small round
colonies

Heamophilus influenza

Expected physical
appearance
red

Chocolate

DCA

Macconkey
agar

Escherichia coli
Proteus mirabilis
Salmonella
typhimurium
Enterococcus
Escherichia coli

Enterococcus
Staphylococcus aureus

Growth inhibited
Lactose fermenter.
Pink colonies
Non-lactose
fermenter. Colourless
and non swarming
Growth inhibited
Growth

Neisseria gonorrhoea
Neisseria meningitiditis
Escherichia coli
Candida albicans

Growth
Growth
Non growth
Creamy colonies

Cryptococcus
Campy jejunii

Mucoid colonies
Grey colonies (1 mm)

Proteus mirabilis

Nutrient
agar
TMA
Sabaroud
dextrose
agar
Campy
agar

Pink colonies
Colourless colonies
Colourless colonies

Streptococcus
eperdimidis
Escherichia coli

Light pink

Pink

Clear
Light chocholate
Cream
transparent.

Growth inhibited
Growth inhibited

Culture maintenance:
This culture maintenance is done for the purpose of future reference. Procedures:
Culture on the plate will be swabbed using a sterile cotton swab
Aseptically transfer into glycerol that had been earlier transferred into cryo
tube (1.0-1.8 mL).
Stir the swab inside the glycerol until it turns cloudy.
Labelled the cod and fill in the record book.
Store in -200C temperature for up to a year.
Recording stock culture:
Example; CRE producer (Carbopenamase producr)
Kliebsella pneumoniae
Escherichia coli
Enterococcus clocae.
Code: X X X
CRE
(code name for m/o)

/ X X X /15/A
(Number according to previous record)

Other stock:
ESBL (Extended spectrum beta-lactamase), Amp C producer
Escherichia coli
Kliebsella pneumoniae
Proteus mirabilis
Group B:

 Streptococcus group B
Group MRSA
Grou BUPS (Burkholderia pseudumallei)

MYCOLOGY LAB.
There are two types of sample
Sterile subculture first on SDA & Mycosel to get yeast or mould
If yeast: subculture on PDA, germ tube and urea test. Undergo API 20C
AUX
Non-sterile subculture first on SDA & Mycosel to get yeast or mould
direct test to gem tube test, urea test. Undergo slide culture (CMT) and
sugar assimilation.
If mould: do slide cultre (PDA) and proceed with LPCB
LPCB: The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used
method of staining and observing fungi and is simple to prepare. The preparation has three
components:

Phenol: kills any live organisms;

Lactic acid : It preserves fungal structures, and
Cotton blue : It stains the chitin in the fungal cell walls.
Lactophenol Cotton Blue Solution is a mounting medium andstaining
agent used in the preparation of slides for microscopic examination of
fungi. Fungal elements are stained intensely blue.
Preparation of lactophenol cotton blue (LPCB) slide mounts
Place a drop of seventy percent alcohol on a clean microscope slide.
Material from cultures of filamentous fungi should be removed using a
stiff inoculating wire not the loop used for manipulations with bacteria
or yeasts.
Flame the wire by holding it upright in the hottest part of the Bunsen
flame, just above the blue cone, until the whole length of the wire
glows red hot.
You must ensure that the inoculating wire has cooled before placing it
in a fungal culture it should have cooled sufficiently after
approximately ten seconds.

Remove the cap from the tube but do not put it on the bench. Kill any
contaminating microorganisms by flaming the neck of the tube.
Remove a small amount of the culture. For fungal cultures, it is often
useful to take a little of the agar medium together with the fungus. In
any case, the material should be disturbed as little as possible when
being transferred to the slide.
Flame the neck of the tube once more and replace the cap.
Immerse the fungal material in the drop of seventy percent alcohol.
This drives out the air trapped between the hyphae.
Tease out the material very gently with mounted needles.
Do not forget to sterilise the inoculating wire and the needles after use
by heating to red heat in a Bunsen flame
Fungal structures are readily visualised after staining with a
lactophenol cotton blue dye preparation.
Before the alcohol dries out add one or at most two drops of the stain.
A common fault is to add too much to the preparation. Holding the
coverslip between your index finger and thumb, touch one edge of the
drop of stain with the edge of the coverslip.
Lower the coverslip gently onto the slide, trying to avoid air bubbles.
Your preparation is now ready for examination.
Make the initial examination using a low power objective lens. The
thinner parts of the preparation, generally around the edges of the
mounted material, will yield the best images.
Switch to a higher power 40X objective for more detailed examination
of spores and other structures.

Germ tube:
Positive geram tube; possibilities:
o Candida albicans
o Candida dubliniensis
o Tropicalis
Positive germ tube for sterile: proceed with API 20C AUX
Positive germ tube for non-sterile: subculture on PDA at 45 oC, if grow, it
indicates the the presence of Candida albicans.
Negative germ tube:
Proceed with sugar assimilation test:
o Take some of the colony on the plate using sterile cotton swab
o Stir well inside 1mL of peptone water to produce about 4-7
mcFarland reading (cloudy)
o Melt bacto agar inside the steamer for a while.
o Once melt, cool down for a bit. Pour into plate and immediately pour
in 1 mL of culture suspension.
o Wait until solidified and add sugger labelled 1-14 that was kept
anhydrously inside fridge.
Numb
er
1
2

Sugar
Glucose
Sucrose

1
6
5

2
7
4

13
12

9
14
11

10

3
4
5
6
7
8
9
10
11
12
13
15

Trehalos
e
Maltose
Galactos
e
Cellbiose
Arabinos
e
Lactose
Melibios
e
Raffinos
e
Rhammo
se
Mannitol
e
Inositole
Xylose

Incubate overnight and observed for the formation of the clear

zone surround the sugar disk. This indicate the inability of the
microorganisms to metabolize certain type of sugar.
Germ tube procedues:
A test tube filled with plasma will added fungal colony.
Incubate for overnight
After that, drop on glass slide and covered with glass slide.
Observed for the presence of chlamydospore which is positive.
UREA TEST
Positive: Trichosporum, Cryptococcus, rhodotorula
Negative: Glabrata, tropicalist, (also negative for germ tube)
Procedure:
Fungul culture will be inoculated by using sterile loop into the medium
Incubate overnight and positive will be red pinkish and negative will stay
yellow.
o

SLIDE CULTURE
Slide culture can be devided into two:
CMT- (Corn meal Agar) yeast.
PDA (Potatoes Dextrose Agar) for mould
Procedures:

Sterile wooden
stock was cut into
half and in V
shape to support
slide culture

Put on glass slide

on the sterile
wooden stick

Using sterelized
dissector, cut in
square shape
(PDA/CMA)

Insert a wet cotton wool to

The square cut agar was
give
humid
environment
place on the slide and
needed
by
the
fungus to
transfer a loopful of culture to
grow. Incubate for 48-72
4
different
side
and
hours
and
observed
immediately cover with a
Aim: to look the present and type of hyphae. Type of hyphae observed
must be tally with sugar assimilation result.
API 20C AUX FINAL TESTING FOR POSITIVE GERM TUBE.
Procedure:
100 microlite sample + distilled water and add with API 20AUX medium.
Transfer into well (1 drop- 50 microlitre)

Incubate for 48-72 hours and observed for cloudy well.

Key in data API AUX web and it will auto interpret.
If result < 80% repeat API 20-C AUX.

INDIAN INK.
Purpose: used in CSF sample to examine the presence of encapsulated yeast.
Cryptococcus neuformans SINCE it lost its ability to produce capsule in artificial
media.
Drop a drop of indian ink and mix with a drop of sample.
Put on cover slide and observed for any capsule microscopically.
SUGAR DISK PREPARATION.
Procedures:
Sterile disks will be place inside petri dish. By using a dropper, drop sugar
solution onto disk.
Incubate overnight to dry.
Store in dry and cold storage.
E-TEST (ANTIFUNGAL SUSCEPTIBILITY TEST FOR YEAST)
Aim: to test for Candida albicans, candida species and crptococcus neofromans.
Inoculom:
Candida species: SAB in 0.85% NaCl= 0.5 macFarland.
Cryptococcus neofromans: SAB in 0.85% NaCl= 1 McFarland.
Procedure:

Dip in a sterile cotton swab to suspension.

Make 3 direction of streaking.
Let it dried for 10-15 minutes before putting E-test strip.
Incubation at 35oC
o 24-48 hours: Candida
o 48-72 hours: Cryptococcus

Quality control
Antifungal

Candida crusei

Amphotericin B
Fluconazole
Voriconazole
Caspofungin
Posaconazole
Interpretation:
Antifungal
Amphoterici
nB
Fluconazole
Voriconazole
Caspofungin
Posaconazol
e

Candida
parapstosis
0.25-1
1-8
0.016-0.064
0.25-2
-

0.5 2.0
128 - >256
0.25-1
0.25-1.0
-

Susceptible

Intermediate

1.0 ug/mL
8.0 ug/mL
1.0 ug/mL
2.0 ug/mL

Candida albicans
0.125-0.5
0.125-0.5
0.004-0.0016
0.064-0.25
-

2.0 ug/mL

Susceptible dosage
dependent
-

Resista
nt
>4.0

1.6 32 ug/ml
3.0 ug/mL
-

>6.4
>4.0
>2.0
-

CRYPTOCOCCUS LATEX AGGLUTINATION

Type of specimens:
CSF: boil for 5 minutes, cool down and centrifuge
Serum: treat with protease > cap and seal tube
Procedures:
Resuspend test latex by inverting several times.
Place a drop in each circle.
Place a drop of supernantant of sample
Mix and spread by using straw.
Place on rotator, if agglutination occur positive
If negative report. If positive, proceed with serial dilution,
Media used in Mycology lab (some were prepared, some were being provided):
Sabauraud dextrose agar
Mycosel agar
Brain heart infusion
Bacto agar
Corn meal agar
Potato dextrose agar
Rose well park memorial inst. Medium
Test for certain species:
Species

Tests

Candida albicans

Candida spp.

Mould

Mould microscope

Germ tube/urease
Subculture on SDA 30 degree
Subculture on SDA- 45 degree
Germ tube/urease
Sugar assimilation and CMT
Resub on SDA
E. TEST/API 20C AUX
LPCB
Cellophane tape techniques
Slide culture on PDA
KOH (10%,20%,30%)
Tissue

VIROLOGY LAB
Samples procedures (processing):
RESPIRATORY SAMPLE; eg: Bronchial washing, nasal, throat washing, aspirates).
There are two types of samples; speciments with excessive mucus and
specimens without excessive mucus, where both will have slightly
difference procedures.
Specimens without excessive
mucus
Resuspend the fljuid to
dislodge.
Transfer 4 drops to eppy tube
containing 2mL medium
Add 100mL of Antibiotic and
fungizole
Mix by swirling bottle,
incubate 1 hour before
inoculation.

URINES SAMPLE:
Processing procxedure:

Specimens with excessive mucus

Dilute sample with PBS

Resuspend the liquid.
Add 2-4 drops inti mediu,
Remaining dilute sample will
be inserted into screw capped
bottle.
Centrifuge 2300 rpm at 5
minutes.
Transfer 2mL inti tube
Add antibiotic and fungizone,
swirl and incubate.

Suspend specimen and instert into tube (2 ml medium)

Add 450 microlitre fungizone and 150 microlitre antibiotic. Mixed well.

BODY FLUID SUCH AS:

Bile (inoculate directly)
CSF (inoculate directly)
Perocardial (inoculate directly)
Pleural (inoculate directly)
If suspect contamination, transfer to tube and add fungizone and antibiotic
SELECTION OF CELL CULTURE FOR VIRAL ISOLATION
Virus to isolate

HEP-2

Respiratory syncytial virus

Adenovirus
Para influenza
Influenza
Herpes simplex
Cytomegalovirus
Enterovirus

/
/
/

Vero

MDC
K

MRC
5

RD

/
/

/
/

/
/
/

Notes: MRC-5 grows at slower rate. Up to 1 week of post seeding are acceptable.
All respiratory specimens should be at least incolulated into HEP2 and MDCK.
INOCULATION OF CELL CULTURE
Procedures:
Determine the cell culture to be inoculated. 2 tubes for each cell
Discard medium from the tube and rinse with 1-2 mL of PBS (to remove
residual serum)
Insert 1 mL of maintenance medium and 2 ug/mL TPCK-Trypsin
Add 0.2 0.3 mL of prepared specimens.
Incubate for 30-40 minutes (5-7 degree slant).
Reefed with maintenance medium and incubate at 35-37 degree in C02
condition with loosely capped.
Observation: cytomegalovirus will be observed for 28 days. Observed
development of CPE, save aliquot of positive at -70 oC.
PROCESS SPECIMENS CHART:
Processed
Inoculated into cell

CPE & cell

die
(5-14

Cell
die
Day 1
Reinoculate
. of
incoluation

Cell
die
Day 1

Contaminati
on
1-4 days

Passage
Reinoculate
to new
(filter
Observation
of
10-14
cell
specimens
days, CMV= 28 days

Extreme
Ph
change
Change
the
medium

CP
E

NO CPE (END OF
INCUBATION)

Harvest
cell
culture

Respirato
ry
virust/CM

IFA
staining

Negati
ve

Positiv
e

Virus
isolated

Other
cells/virus/HS
V

CULTURE MAINTENANCE AND PROCESSING

REFEEDING
Refeed with fresh culture maintenance at 4-5 days after inoculation and culture
that exhibit high pH changes.
Procedures:
Discard medium in hyphochlorite solution. Leave 0.2/0.3 Ml
Add around 1 mL of culture maintenancemedium
Reincubate and continue observation under microscope.
SUBPASSAGE
Aim: for culture that went destructive within day 1-4.
Procedure:
Select cell culture and identify tube to be subpassaged,
Scrapped the cell from the surface to mix.
Deliver 0.1 mL of harvested culture to tube. strong CPE will be diluted in
VTM.
Reincubate
REINOCULATION
Aim: for contamination that occurs during day 1-4.
Procedures:
Select suitable medium.

Identify first sample to be inoculated. In case of contamination, filter the

primary sample through syringe.
In case of toxicity, use only half volume.
Deliver 0.2/0.3 mL of processed sample.
Incubate and continue observation.

ISOLATE, HARVESTING, IDENT AND STORAGE.

Harvest isolates for identification when:
CPE occurs.
Cell culture die after 1 day
No CPE at the end of incubation.
Procedures:
Select culture for harvesting.
Scrap cell from the culture tube and mixed by pipetting up and down
Transfer 0.5 mL of harvested material into 2 mL of eppy tube.
Centrifuge 1500 rpm for 15 minutes.
Discard supernatant and resuspend cell pallet in PBS (1ML)
Centrifuge and discard supernatant.
Resuspend 0.3/0.5 mL PBS.
Deliver 25 microlitre of sample to Teflon coated slide. Let air dry.
Fix by dipping into aceton, dry and perform staining.
If positive, identify original culture tube to be frozen.

DFA STAINING
Procedures:
Identify the type of sample and mix by resuspend using pipette
Add 4-5 drops of fresh culture into virus transport medium, and add 300
microlitre of fungizone and 100 microlite of antibiotic.
The remaining fresh culture will be transferred to test tube ( the volume
added depends on the thickness of the sample. Add some PBS buffer and
mix well.
Centrifuge at 2300 rpm for 5 minutes. Discard the supernatant, add some
PBS (make sure it is not too thick) and transfer all into eppy tube. add o.5
microlitre of buffer into eppy tube.
Fill the wells with 25 microlitre of sample each. Fill positive controls.
Aim: to make sure the technique used is right.
Fix in acetone for 10 minutes. After 10 minutes, let it dry for a while and
add 50 microlite of DFA stain, and spread using pipette tip without
touching the sample.
Incubate in moist chamber for 15 minutes.
After incubation, wash thoroughly with PBS, before dipping into PBS I &
PBS II
Blow until completely dry.
Put mounting liquid and put on cover slide.
Observed under UV microscope. Green apple cell is a positive cell infected
by virus.
Procedd with full identification.

# in full ident, repeat all above steps except using different staining.