INOCULATION

Dr. Sadia Ikram

INOCULATION OF CULTURE
IN VARIOUS MICOBIOLOGICAL
SPECIMENS
WHAT IS INOCULATION AND
WHY IS IT REQUIRED
Inoculation is the means of growing &
isolating bacteria on artificial medium.



HOW TO MAKE WIRE LOOPS
Nichrome wire for making wire loop.
Length of wire loop should be 6cm.
Diameter should be 2mm.
Disposable wire loops may also be used,
but they are expensive.



WIRE LOOPS
REQUIREMENTS FOR INOCULATION
Disposable gloves.
Flame sterilizer.
Wire loops.
Racks for holding specimens.
Pipettes or disposable syringes.
Waste container with hypochlorite.
Media.
Centrifuge.
STERILIZATION OF WIRE LOOPS
INOCULATION IN PETRI DISHES
Also called as plating out, or looping out.
There can be:
1. Full plate inoculation .
2. Half plate inoculation.
3. One third plate inoculation.
BEFORE INOCULATION
Bring the petri dishes at room temperature by putting in
incubator.
Lid aside in incubator.

INOCULATION
Technique used must provide :
single colonies for identification.
must show if culture is pure or mixed.

METHOD
HOW TO INOCULATE
FULL PLATE INOCULATION
HALF PLATE INOCULATION
ONE THIRD PLATE INOCULATION

INOCULATION OF SLOPES
METHOD:
To innoculate slopes e.g. loffler serum,
use a sterile straight wire to streak the
innoculum down the centre of slope and
then spread the innoculum in zig zag
pattern.
To innoculate slope and butt e.g. KIA,
use a sterile straight wire to stab in to butt
first and then use the same wire to
streak the slope in zig zag pattern.
INOCULATION OF STAB MEDIA
METHOD:
Use a sterile straight wire loop, and stab
the centre of medium, and then withdraw
the wire loop along the line of innoculum.
INOCULATION OF FLUID MEDIA
INOCULATION OF TIPS
METHOD
First put the tip in normal saline.
Inoculate on blood and macconkey agar.
Blood agar Macconkey agar
INCUBATE
INOCULATION OF VARIOUS
SPECIMENS

1.BLOOD/BONE MARROW
Brain heart infusion broth
or
commercial media- bactec
INCUBATE
and
EXAMINE
after 24, 48
,72h--7 days,
For turbidity/
Subculture
on


BLOOD AGAR MACCONKEY AGAR
INCUBATE
2.SPUTUM
Immediate processing
Gram stain : <10 polys:1 sq epith cells
saliva not fit for culture
Culture on

BLOOD CHOCOLATE MACCONKEY
INCUBATE IN CARBON DIOXIDE
JAR
Note;If request for M.tuberculosis comes,
Z-N staining
LJ medium for inoculation

3.THROAT AND MOUTH SPECIMENS

Culture on


BLOOD
AGAR
INCUBATE
Gram stain
If request for diphtheria comes
do
ALBERT STAINING
Culture on TELLURITE BLOOD AGAR


Culture on


Incubate chocolate
agar in candle jar
BLOOD CHOCOLATE
Gram stain
4. NASAL / NASOPHARYNGEAL
SWABS
BRONCHIO ALVEOLAR LAVAGE (BAL)
Collection Bronchoscopy
Quantitative culture 0.01 ml on
Blood MacConkey Chocolate
Cut off > 10
5
Cfu /ml

Gram stain : 1 organism / oil field

Culture on

BLOOD MACCONKEY CHOCOLATE
INCUBATE
Chocolate agar
in candle jar
5. EAR DISCHARGES
In case of chronic ear discharge
put in
ANAEROBIC JAR





In case of fungal infection, culture
on sabourad media.
Gram smear


6.EYE SPECIMEN
Geimsa smear for chlamydia
trachomatis
Culture on










THYER MARTIN
Medium for culture
if Neisseria gonorrhea
is suspected in a
newborn, infant.
Incubate
BLOOD CHOCOLATE
7. SKIN SPCIMENS

KOH preparation if fungus is suspected.
Culture on






SABOURAD
For fungal
culture





TELLURITE
If diphtheria
suspected
BLOOD MACCONKEY
INCUBATE
Gram stain
8. PUS SPECIMENS
Culture on


Blood
+
neomycin




PUT IN ANAEROBIC
JAR IF ANAEROBES
ARE SUSPECTED.
BLOOD MACCONKEY
ROBERTSON
COOKED MEAT
MEDIA
Sub
Culture
on
Gram smear
9.UROGENITAL SPECIMENS
INCLUDE
urethral specimens.
Vaginal specimens.
Cervical specimens.
Genital specimens.
Gram smear -pus cells/ bacteria.
intracellular diplococci (gonorrhea)
yeast cells (vaginitis)
Godenella(gram neg cocco bacilli)
Haemophilus duccri (gram neg bacilli)
BIPOLAR STAINING

CULTURE on Anaerobic blood
Thayer martin media Blood agar Macconkey agar agar
For neisseria
gonorrhea
INCUBATE
10. FAECAL SPECIMEN
Put the specimen in selenite F broth
for 6 hours
&
Sub culture
on

Macconkey agar XLD/DCA
INCUBATE
IF CHOLERA is suspected culture
on
TCBS AGAR


DIRECT MICROSCOPY- For VIbrio cholera
motility and Amoeba with ingested RBcs in
dysentery

INCUBATE
11. URINE
Deal immediately /if delay refrigerate the sample.

PHYSICAL
EXAMINATION
CULTURE on
IF CALIBRATED LOOP IS USED
IF STRIP IS USED
INCUBATE
BLOOD MACCONKEY CLED
For carriers of S.Typhi
INCUBATE the sample for 6 hours in
SELENITE F BROTH



SUB CULTURE
on


MACCONKEY
AGAR
BLOOD
AGAR
INCUBATE
12.FLUID / EFFUSIONS
Physical Examination
Centrifuge
10 ml
sediment
for
culture
Gram and Z-N

OR
5-10
ml


CULTURE on
Blood Macconkey Chocolate
5-10%CO2
INCUBATE
IN CANDLE
JAR
Gram stain
Zn stain
Microscopy -- look for uric acid crystals
if joint fluid comes.




In case request for T.B comes, process
for tuberculosis.
13. SEMEN

Physical
Examination

CULTURE on

Blood Macconkey Chocolate

INCUBATE in




CANDLE JAR
for chocolate
agar


Physical
Appearance



CENTRIFUGE the sample, separate
The SEDIMENT for processing

14. CSF
Deal immediately/ If delay
incubate at 35 c


SEDIMENT
USED FOR
CULTURE
on
BLOOD MACCONKEY CHOCOLATE


Incubate chocolate
agar in candle jar
Gram stain/Zn stain if request for chronic
meningitis comes.
(Immediate reporting on telephone)
Indian ink preparation for Cryptococcus
neoformans.



Wet preparation _ for Amoebae.
HANDLING ANAEROBES
Appropriate specimen
pus in syringe without
aircolumn or
In Robertsons cooked
meat medium
Note swabs are unfit

INCUBATE
in anaerobic jar

ANAEROBIC CULTURE
Culture all specimen of the day in one batch to consume
only one anaerobic kit.
Culture direct pus or sub culture from RCM on blood
agar or Neomycin blood agar.
Immediately put in anaerobic jar and culture on one plate
pseudomonas as anaerobic control and insert the
anaerobic sachet after putting 10ml tap water and close
the lid tightly.
Catalyst under the lid should be heated red hot each
time before use.
CONCLUSION
AS YOU SOW, SO SHALL YOU
REAP