Contents

Introduction to work flow

Aseptic / sterile techniques
Microbiology Techniques
Controls

Work flow @ General Microbiology

Lab

Sample
reception
@ Sample
cell

Acknowled
gement of
the sample
in the
system
(Ackn)

Sample
sent to the
lab for
analysis
with RoA
(Request of
analysis)

Sample
reception
in lab

Sample
preparation

Analysis is
continued

Analysis workflow

Confirmation
Plates marked for analysis
traceability

Sample
preparation

Result
Reading

Dilutions &
Plating

2.5 cm
Plates are stacked in a
stack of 6 petriplates .

Result
Recording

Training General Microbiology

Laboratory

Microorganisms analyzed in NQAC

Pathogenic

Salmonella
Shigella
Vibrio cholerae
Vibrio parahaemolyticus
Clostridium perfringens
Clostridium botulinum
Listeria monocytogenes
Legionella
All the parameters are analysed
using particular LI (Laboratory
Instructions) which are formulated
using ISO at NRC (Neste research
centre) .
In few cases NRC recommends to
ISO. (In theses cases we directly
follow the ISO)
9/16/2015

Non Pathogenic

Aerobic mesophilic germs

Burri test (Micrococci)
Coliforms
Sulphite reducing anaerobic spores
S.aureus
B.cereus
Bacterial Spores
Enterobacteriaceae
E.Coli
Lactic Acid Bacteria
Howard mold count
Faecal Streptococci
Flat Sour organisms
Yeast & Spores
Yeast and Mold

CQAL Moga

Why Do We Test For Bacteria In Foods?

Foodborne pathogens
Why? Pathogens cause illness!
Challenges for pathogen detection:
Heterogeneous distribution in a lot or even a sample.
Pathogen cells on products are often at low levels and stressed.
Indicator bacteria
Why?
- Easier to detect and quantify for process
control purposes

Aseptic Technique
protective clothing
hand washing
bench cleaning
loop flaming
pipettors

Disinfecting workbenches
with 70% alcohol

Sterile Technique
Microbes in the laboratory can be found all around you, but
they are so small that you cannot see them. Microbes are in
the air, on benches and equipment and in water. They are one
of the major cause of the contamination of samples.
When culturing bacteria or other microorganisms, it is
important to keep your work area as clean as possible.
This prevents the introduction of other microorganisms from
the environment into your culture.
The techniques used to prevent contamination are referred to
as sterile or Aseptic techniques.

Sterile Technique
1. Start by washing your down your work
or lab benches with a surface
disinfectant. The most commonly used
disinfectants for lab use are:
1. 70% Ethanol
2. 200ppm Chlorine solution for
workbenches
3. 1000ppm chlorine solution for floors.

Use clean lab

coat

Why using Disinfectants ??

Ethanol

are most effective when combined with purified water to

facilitate diffusion through the cell membrane; 100% alcohol typically
denatures only external membrane proteins. A mixture of 70% ethanol
diluted in water is effective against a wide spectrum of bacteria. Ethanol
kills organisms by denaturing their proteins and dissolving their lipids and
is effective against most bacteria and fungi, and many viruses, but is
ineffective against bacterial spores.

Ppm solution of chlorine Chlorine is a strong oxidizer.

Sodium hypochlorite act as Oxidizing agent by oxidizing the cell membrane

of microorganisms, which results in a loss of structure and leads to cell
lysis and death.

Sterile Technique (2)

2. Turn off any forced air heating or air conditioning
units that create strong air current in your work
area.
3. A small room or closet that can be closed off is
worth the effort to set-up if you will be doing a lot
of microbial culturing.
4. You can install a UV bulb in a fluorescent light
fixture to surface sterilize your work bench if you
have an enclosed area. Remember to leave the
area when you turn on the UV light source!

Sterile Technique (3)

5. All glassware should be cleaned and
sterilized before you begin.
6. All pipettes, spatulas, and test tube (culture)
racks should also be sterilized.
7. We use sterile, disposable culture tubes,
petri dishes to minimize the quantity of
glassware that we have to sterilize.

Sterile Technique (4)

8. Dont forget to wash you hands after you
finish cleaning and put on a pair of sterile
disposable gloves before you begin.
9. Once your work area is clean, your hands
are clean, and your glassware is clean and
sterile, dont contaminate the work area by
placing dirty items such as pencils, pens,
notes, or books in the sterile work area.

Microbial
Techniques

Preparing Agar Plates

Line your sterile petri plates along the edge of the table. Transfer hot media to a
small sterile container and pour 15-20 ml of the plate media into each petri plate.
The petri plate lid should be open slightly, but not completely open as this
increases contamination. The thermal current created by the hot media prevents
bacteria and fungal spores from landing in your clean dish.

Storage of Agar Plates

1.

Store agar plates upside down. Stack the plates in their original bags for
further protection from contamination.
2. Store agar plates in a refrigerator. Most bacteria cannot grow well in cold
temperatures.
3. Store plates in a cold room if a refrigerator is not available. If you are storing
plates in a cold room, check the plates for condensation a few hours after
pouring. Condensation results from exposure to a heat source that drives
water out of the water and into the lid of the plate. This will dry the agar out
and render it unusable. Turn the plates over if condensation is visible and
monitor closely for more condensation development.
Before using the plates, examine them carefully for microbial growth (tiny colonies
of microbes) that may have grown during storage.
Check for cracking of the agar medium, which indicates that the plates are drying
out. If the plates are not dried out and have not been contaminated, the plates can
be used.

Inoculation of Liquid and

Solid (Slant) Culture Tubes
Step 1: Remove the culture tube stopper or cap
with one (do not set it down) and flame the
mouth of the tube to surface sterilize the
mouth. The heated tube surface will
generate a thermal current that prevents
contamination of the culture.

Inoculation of Liquid and

Solid (Slant) Culture Tubes
Step 2: Without setting any of the culture materials on
the bench, place the sterile inoculation loop in the
culture.
Step 3: Replace cap on the culture tube with the active
microbes and put it in the test tube rack.
Step 4: Without setting the loop down, pick-up a sterile
fresh culture tube with media with one hand, and
remove the cap with the other hand.

Inoculation of Liquid and

Solid (Slant) Culture Tubes
Step 5: Flame the mouth of the clean culture tube.
Step 6: Place the inoculation loop containing the
microbes in the fresh media and swirl the loop in
the loop in the media to ensure even dispersal in
the media.
Step 7: If using a solid media slant tube, follow steps 15 and then zig-zag the inoculation loop across the
slanted surface of the solid media in the tube.

Inoculation of Liquid and

Solid (Slant) Culture Tubes
Step 8: Flame the mouth of the newly inoculated
culture tube and replace the cap.
Step 9: Place the culture tube in test tube rack.
Step 10: Repeat until all of the sterile tubes have been
inoculated. Use a fresh disposable culture loop for
each tube or flame the metal loop after each tube
has been inoculated.

Inoculation of Liquid and

Solid (Slant) Culture Tubes
Step 11: Incubate the culture at the recommended
temperature (check with your supplier for growth
requirements). If using environmental samples,
incubation at room temperature will avoid the
accidental culture of human pathogens.
Step 12: Dispose of all culture materials in a biohazard
bag and sterilize all old cultures before pouring out
cultures and washing culture tubes. Disposable
culture dishes should be melted in an autoclave or
pressure cooker prior to disposal.

Stab Culture
By puncturing a
suitable medium with
a long, straight
charged wire.
For gelatin
liquefaction, stock
cultures & motility

Broth Culture
Inoculated by a
charged loop, pipette
or syringes.
For blood cultures &
sterility testing.

05.10.08

Dr Ekta, Microbiology

Pour Plate Method

1 ml of appropriately diluted
inoculum is added to 15 ml of
molten agar and poured on
petridish.
Colonies appear through out
the depth of medium.
Used to estimate viable count

Inoculating Petri Plates

Step 1:Remove the culture tube stopper or cap with
one (do not set it down) and flame the mouth of
the tube to surface sterilize the mouth. The
heated tube surface will generate a thermal
current that prevents contamination of the
culture.
Step 2: Without setting any of the culture materials
on the bench, place the sterile inoculation loop in
the culture.
Step 3: Replace cap on the culture tube with the
active microbes and put it in the test tube rack.

Flaming & Streaking

Two types of streaking are used on agar plates .
Quadrant streak
Control streak (16 streak)

Quadrant streak

Flaming of the
loop Till it
becomes red hot

16 Streak

Stabbing( on the
butt) & streaking (on
the slat) is done on
agar slats

Serial Dilution of Cultures

Serial dilution techniques should be used in the estimation of
microbial population sizes.
Serial dilution involves the use of a known amount (in ml or
l) in a known volume of liquid media.
A one in ten dilution is made in a new liquid culture tube, and
this process is usually repeated several times. The resulting
cultures are dilutions of 1/10, 1/100, 1/1000, 1/10,000, for
example, of the original sample.
These cultures are plated on petri plates and incubated at the
recommended temperature.

Dilution preparation

We get lesser
count as we
move to higher
dilutions

Counting colonies by
using colony counter

Addition of TTC colors the colonies

& hence facilitate counting

Controls
The samples are analysed by controlling all the
factors involved. To ensure this, four different
controls are set. These are:
AC: Agar control. Only the P+M media is
poured to ensure its sterility. If growth is
observed in this control, the media was
contaminated and hence this means the
growth obtained in other sample plates is not
solely of the sample but of the media too.

 TWC: Tryptone water control. Without inoculating it

with sample, 1 ml of ringer is cultured in the media
and grown. Any growth shows contamination in the
ringer solution used.
Airtest: Only media is poured and set. This plate is
then left open at the workplace for 15 min to test the
sterility of air surrounding the sampling process.
PC: Plate control. Ringer solution is poured in a plate,
and then from that plate 1 ml of ringer is taken and
cultured in another plate. This tests the plate
sterility.