MICROBIOLOGY DIVISION

FOOD QUALITY AND MONITORING LABORATORY

TRAINING
REPORT

ABEL JACOB THOMAS JITHU MATHEWS EAPEN LIBIYA N

POOJA S MOHAMMED ROSHAN N. S SALAMATH I
SETHULEKSHMI V. R
 INTRODUCTION

The NABL accredited Food Quality Monitoring Laboratory (FQML) mainly

has microbiological and chemical divisions for the analysis of various food
samples and it comes directly under the Council for Food Research and
Development (CFRD). Variety of food products from different food
industries like supplyco, private sectors, entrepreneurs and individuals are
analyzed for various microbiological parameters.

Microbiological analysis of Escherichia coli, Staphylococcus aureus,

Salmonella etc. are mainly carried out here. The microbiology lab contains
equipments such as incubator, laminar air flow, biosafety cabinet, autoclave,
automatic TPC machine (TEMPO) etc. for the analysis. This training is
mainly aimed to develop an in-hand experience in a food microbiology
laboratory.
 Microbiology Lab Practices and Safety Rules

1.Wash your hands with disinfectant soap when you arrive at the lab and again
before you leave.
2.Absolutely no food, drinks, chewing gum, or smoking is allowed in the
laboratory. Do not put anything in your mouth such as pencils, pens, labels, or
fingers. Do not store food in areas where microorganisms are stored.
3.Keep your workspace free of all unnecessary materials. Backpacks, purses, and
coats should be placed in the cubbyholes by the front door of the lab. Place
needed items on the floor near your feet, but not in the aisle.
4.Disinfect work areas before and after use with 70% ethanol or fresh 10%
bleach. Laboratory equipment and work surfaces should be decontaminated
with an appropriate disinfectant on a routine basis, and especially after spills,
splashes, or other contamination.
5.Label everything clearly.
6.Replace caps on reagents, solution bottles, and bacterial cultures. Do not open
Petri dishes in the lab unless absolutely necessary.
7.Inoculating loops and needles should be flame sterilized in a Bunsen burner
before you lay them down.
8.Turn off Bunsen burners when not is use. Long hair must be restrained if
Bunsen burners are in use. 11. When you flame sterilize with alcohol, be sure
that you do not have any papers under you.
9.Treat all microorganisms as potential pathogens. Use appropriate care and do
not take cultures out of the laboratory.
10.Wear disposable gloves when working with potentially infectious microbes or
samples If you are working with a sample that may contain a pathogen, then be
extremely careful to use good bacteriological technique.
11.Sterilize equipment and materials.
12.Never pipette by mouth. Use a pipetting aid or adjustable volume pipettors.
13.Consider everything a biohazard. Do not pour anything down the sink.
Autoclave liquids and broth cultures to sterilize them before discarding. waste
material in a biohazard bag and autoclave it before discarding in the regular
trash.
14.Dispose of broken glass in the broken glass container.
15. Report all injuries or accidents immediately to the instructor, no matter how
small they seem.
 EQUIPMENTS

1.LAMINAR AIR FLOW

 A laminar air flow/hood/cabinet is an enclosed workstation that is used to create
a contamination free environment through filters to capture all the particles
entering the cabinet.
 HEPA Filters – High Efficiency Particulate Air Filter is present within the
cabinet that makes the environment more sterile for operation.
 The prefiltered air passes through the filters which trap fungi, bacteria and other
dust particles.
 The filters ensure a sterile condition inside the cabinet, thus reducing the
chances of contamination.

PRINCIPLE – It is based on the laminar flow of air through the cabinet.

The device works by the use of inwards flow of air through one or more HEPA
filters to create a particulate-free environment. The air is taken through a
filtration system and then exhausted across the work surface as a part of the
laminar flow of the air. The air first passes through the filter pad or pre-filter
that allows a streamline flow of air into the cabinet. Next, the blower or fan
directs the air towards the HEPA filters. The HEPA filters then trap the
bacteria, fungi and other particulate materials so that the air moving out of it is
particulate-free air. Some of the effluent air then passes through perforation
present at the bottom rear end of the cabinet, but most of it passes over the
working bench while coming out of the cabinet towards the face of the
operator. The laminar flow hood is enclosed on the sides, and constant positive
air pressure is maintained to prevent the intrusion of contaminated external air
into the cabinet.
2.BIO-SAFETY CABINET
Biosafety cabinets or Biological Safety Cabinets (BSCs) are also called
Microbiological Safety Cabinets. These enclosed containments feature a well-
ventilated hood and are intended to safeguard personnel, products, and the
environment from hazardous particulates and infectious agents.

BSCs offer three levels of protection: Personal, Sample, Lab/Environment.

3.AUTOCLAVE
 An autoclave or steam sterilizer is a machine that provides a physical method of
sterilization by killing bacteria, viruses, and even spores present in the material
put inside of the vessel using steam under pressure.
 Autoclave sterilizes the materials by heating them up to a particular temperature
for a specific period of time.

PRINCIPLE - The autoclave works on the principle of moist heat sterilization

where steam under pressure is used to sterilize the material present inside the
chamber. The high pressure increases the boiling point of water and thus helps
achieve a higher temperature for sterilization. Water usually boils at 100°C
under normal atmospheric pressure (760 mm of Hg); however, the boiling
point of water increases if the pressure is to be increased. Similarly, the high
pressure also facilitates the rapid penetration of heat into deeper parts of the
material, and moisture present in the steam causes the coagulation of proteins
causing an irreversible loss of function and activity of microbes. This principle
is employed in an autoclave where the water boils at 121°C at the pressure of
15 psi or 775 mm of Hg. When this steam comes in contact with the surface, it
kills the microbes by giving off latent heat. The condensed liquid ensures the
 moist killing of the microbes. Once the sterilization phase is completed (which
depends on the level of contamination of material inside), the pressure is
released from the inside of the chamber through the whistle. The pressure
inside the chamber is then restored back to the ambient pressure while the
components inside remain hot for some time.

4.HOT AIR OVEN

 A hot air oven or forced air circulating oven is a laboratory instrument that uses
dry heat to sterilize laboratory equipment and other materials. To destroy
microorganisms and bacterial spores, a hot air oven provides extremely high
temperatures over several hours.
 The widely used temperature-time relationship in hot air ovens to destroy
microorganisms are 170 degrees Celsius for 30 minutes, 160 degrees Celsius
for 60 minutes, and 150 degrees Celsius for 150 minutes. The temperature
range of a hot air oven is 50 to 300°C. It can be controlled by using a
temperature regulator.

PRINCIPLE - Sterilization by dry heat is performed by conduction. The

temperature is consumed by the surface of the objects, then moves towards the
core of the object, coating by coating. The whole object will ultimately attain
the temperature needed for sterilization to take place. Dry heat causes most of
the injury by oxidizing particles. The primary cell components are damaged
and the organism dies. The temperature is kept for about an hour to eliminate
the most ambitious of the resistant spores.

5.INCUBATOR
 An incubator, in microbiology, is an insulated and enclosed device that provides
an optimal condition of temperature, humidity, and other environmental
 conditions required for the growth of organisms. It is necessary for cultivating
microorganisms under artificial conditions.
 An incubator can be used to cultivate both unicellular and multicellular
organisms.

PRINCIPLE - An incubator is based on the principle that microorganisms

require a particular set of parameters for their growth and development. All
incubators are based on the concept that when organisms are provided with the
optimal condition of temperature, humidity, oxygen, and carbon dioxide
levels, they grow and divide to form more organisms.

6.INOCULATION LOOP
 An inoculation loop (also called a smear loop, inoculation wand or
microstreaker) is a simple tool used mainly by microbiologists to pick up and
transfer a small sample of microorganisms called inoculum from a microbial
culture, e.g., for streaking on a culture plate. This process is called inoculation.

7.INCOLUATION WIRE/NEEDLE
 An inoculation needle is a laboratory equipment used in the field of
microbiology to transfer and inoculate living microorganisms. It is used
primarily for studying bacteria and fungi on semi-solid media.
 TOTAL VIABLE COUNT

 Total viable count (TVC), gives a quantitative estimate of the concentration of

microorganisms such as bacteria, yeast or mould spores in a sample. The count
represents the number of colony forming units (cfu) per g (or per ml) of the
sample.
 A TVC is achieved by plating serial tenfold dilutions of the sample until
between 30 and 300 colonies can be counted on a single plate. The reported
count is the number of colonies counted multiplied by the dilution used for the
counted plate
 A high TVC count indicates a high concentration of micro-organisms which
may indicate poor quality for drinking water or foodstuff.
 In food microbiology it is used as a benchmark for the evaluation of the shelf-
life of foodstuffs.

PROCEDURE –

 Aseptically add 50g of sample to 450ml Butterfield's buffer to make a dilution

of 10-1. If sample is suspected to have a higher microbial load, go for higher
dilutions of 10-2, 10-3… by transferring 10ml of sample from 10 -1 dilution to
(90ml) 10-2 and so on.
 Shake all dilutions vigorously 25 times in 7s. Not more than 15 min should
elapse from the time sample is blended until all dilutions are in appropriate
media.
 Pipette 1ml of each dilution into separate, duplicate, appropriately marked Petri
dishes. Reshake dilution bottle 25 times in 30 cm arc within 7s if it stands
more than 3 min before it is pipette into Petri dish. Add 12-15 ml plate count
agar (cooled to 45 ± 1°C) to each plate within 15 min of original dilution. For
milk samples, pour an agar control, pour a dilution. water control and pipette
water for a pipette control. Add agar to the latter two for each series of
samples. Add agar immediately to Petri dishes when sample diluents contain
hygroscopic materials, e.g., flour and starch. Pour agar and dilution water
control plates for each series of samples. Immediately mix sample dilutions
and agar medium thoroughly and uniformly by alternate rotation and back-
and-forth motion of plates on flat level surface. Let agar solidify. Inverts
solidified Petri dishes, and incubate promptly for 48 ± 2 h at 35°C. Do not
stack plates when pouring agar or when agar is solidifying.

N= ƩC

[n1 + (0.1 * n2) * d]

RESULT –

CALCULATED
SAMPLE APC
COUNT

B
 CONCLUSION

After undergoing one week training at Microbiology division of Food Quality

Monitoring Laboratory at CFRD, we got to know a lot of new techniques and
information on how a microbiology laboratory analyses food samples, starting
from the receiving of the sample and also the confidentiality of the received
sample. Mrs. Veena was so kind and helpful in explaining even the basic things
and therefore we take this opportunity to thank her for her support throughout
the training period. It was such a great experience to come across the staffs of
FQML, where they also played their part by sharing their knowledge. This
training program will be of great help and will surely contribute to the in hand
practical knowledge personally. We were trained on taking the Total Plate
Count of Green Chilly powder and also in preparing media such as PCA,
BGLB, Lactose Broth, Mac Conkey Broth etc.

We thank Smt. Grace Baby for her guidance and support throughout the training
work and also all other staffs of microbiology division of FQML for their co-
operation.