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Contents
Contents
• Introduction to work flow • Aseptic / sterile techniques • Microbiology Techniques • Controls
Introduction to work flow
Aseptic / sterile techniques
Microbiology Techniques
Controls

Work flow @ General Microbiology Lab

Acknowled Sample gement of reception Sample sent to the lab for analysis with RoA (Request
Acknowled
Sample
gement of
reception
Sample
sent to the
lab for
analysis
with RoA
(Request of
analysis)
Sample
the sample
Sample
Analysis is
reception
@ Sample
in the
preparation
continued
in lab
cell
system
(Ackn)
Analysis workflow
Analysis workflow
Result Recording Result Reading Confirmation Plates marked for analysis traceability Dilutions & Plating
Result
Recording
Result
Reading
Confirmation
Plates marked for analysis
traceability
Dilutions &
Plating
Sample
preparation
2.5 cm
Plates are stacked in a
stack of 6 petriplates .
Training – General Microbiology Laboratory
Training – General Microbiology
Laboratory
Microorganisms analyzed in NQAC Pathogenic Non Pathogenic Salmonella Shigella Vibrio cholerae Vibrio
Microorganisms analyzed in NQAC
Pathogenic
Non 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)
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
Why Do We Test For Bacteria In Foods?
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
Aseptic Technique

…protective clothing …hand washing …bench cleaning …loop flaming …pipettors

Disinfecting workbenches with 70% alcohol
Disinfecting workbenches
with 70% alcohol
Sterile Technique
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.

into your culture.  The techniques used to prevent contamination are referred to as sterile or
Sterile Technique
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:

Use clean lab coat

1. 70% Ethanol

2. 200ppm Chlorine solution for workbenches

3. 1000ppm chlorine solution for floors.

Why using Disinfectants ??
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)
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)
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)
Sterile Technique (4)

8. Don’t 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, don’t contaminate the work area by placing “dirty items” such as pencils, pens, notes, or books in the sterile work area.

contaminate the work area by placing “dirty items” such as pencils, pens, notes, or books in
Microbial Techniques
Microbial
Techniques
Preparing Agar Plates
Preparing Agar Plates
Line your sterile petri plates along the edge of the table. Transfer hot media to
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
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
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
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
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 1- 5 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
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
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
Stab Culture

By puncturing a suitable medium with a long, straight charged wire.

For gelatin liquefaction, stock cultures & motility

a suitable medium with a long, straight charged wire. • For gelatin liquefaction, stock cultures &
Broth Culture
Broth Culture

Inoculated by a charged loop, pipette or syringes.

For blood cultures & sterility testing.

by a charged loop, pipette or syringes. • For blood cultures & sterility testing. 05.10.08 Dr
Pour Plate Method
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

agar and poured on petridish. • Colonies appear through out the depth of medium. • Used
Inoculating Petri Plates
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 . 
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
Flaming of the
loop – Till it
becomes red hot
streak Flaming of the loop – Till it becomes red hot 16 Streak Stabbing( on the
16 Streak
16 Streak

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

Serial Dilution of Cultures
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 μl) 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
Dilution preparation
We get lesser count as we move to higher dilutions Counting colonies by using colony
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
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.

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