A brief description of laboratory aspects of Food Microbiology

2. LABORATORY DESIGN
AND
EQUIPMENT

3. MAIN OBJECTIVE BEHIND DESIGNING A
LABORATORY
 The primary objective in laboratory design should be to
provide a safe, accessible environment for laboratory
personnel to conduct their work.
 A secondary objective is to allow for maximum
flexibility for safe research and teaching use. Therefore,
health and safety hazards shall be anticipated and
carefully evaluated so that protective measures can be
incorporated into the design wherever possible.

4. Standards required for designing a laboratory
Employees should be protected from exposure to substances and biological agents that may
be hazardous to health.
The advisory Committee on Dangerous pathogens (ACDP), UK defined four hazardous grou
for biological agents:
1. Group 1: a biological agent that is unlikely to cause human disease. Eg. Lactobacillus spp.
Saccharomyces spp.
1. Group 2: a biological agent that can cause human disease and may be hazard to employee
it is unlikely to spread to the community and there is effective prophylaxis or effective
treatment available. Eg. B. Cereus, Clostridium spp., Enterobacter spp., E.coli (Non-toxige
Klebsielaa spp., L. monocytogenes, Salmonella, S. aureus, Streptococcus spp.
3. Group 3: Can cause severe human disease and may be a serious hazard to employees;
it may spread to the community, but there is effective prophylaxis or treatment available
eg. Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei,
Ehrlichia sennetsu (Rickettsia sennetsu), Escherichia coli, verocytotoxigenic strains
(eg O157:H7 or O103), Mycobacterium africanum, Mycobacterium tuberculosis ,
Salmonella typhi, Shigella dysenteriae
4. Group 4: Causes severe human disease and is a serious hazard to employees; it is likely to
spread to the community and there is usually no effective prophylaxis or treatment availa
Eg. very infectious viruses such as Ebola virus and Lassa fever viruses

5. The most common basic microbiology tests carried out in most food
industry laboratories are the TCC and enumeration tests for coliforms
yeasts and moulds and S. aureus.
However, some Hazard group 2 organisms will occasionally
occur on the plates, especially if the samples examined are
unprocessed raw food materials.
Therefore, for practical purposes, food microbiology laboratory desig
and construction takes account of the guidance given by ACDP for th
containment of Hazard Group 2 organisms.
ORGANISMS PREVALENT IN FOOD MICROBIOLOG
LABORATORY

6. Containment levels required for organisms in Hazard Group 2 affecting laboratory s
and equipment, adapted from The Advisory Committee on Dangerous Pathogens (A
1. The laboratory should be properly ventilated and engineering controls and working
arrangements must be devised so as to counter the risk of air borne transmission to othe
areas.
2. When undertaking procedures that are likely to give rise to infectious aerosols, a Class I
microbiological safety cabinet should be used.
3. There must be safe storage of biological agents (microorganisms).
4. An autoclave for the sterilisation of waste materials should be readily accessible in the
same building as the laboratory, preferably in the laboratory suite.
5. Materials for autoclaving should be transported to the autoclave in robust and leak proof
containers without spillage.
6. There should be means for the safe collection, storage and disposal of contaminated was

7. Biosafety cabinets are used to provide primary containment in the
laboratory when the investigator is using potentially infectious
materials.
But which direction should the laminar flowmove: vertically
(downward from a filter positioned above the work surface) or
horizontally (forward from a filter positioned behind the work
surface)?
1. Clearance requirements,
2. Process location,
3. Work surface design, and
4. Operator safety
In vertical laminar flow hoods, fan/filter units are typically
positioned in the ceiling. By directing the laminar flow downward,
vertical laminar flow reinforces the effect of gravity and sweeps
particles out of the enclosure. Micro-contaminants may not have
substantial mass, but most particles do eventually settle on a work
surface or the floor of a room, and vertical low helps get them there
faster.

8. Vertical laminar air flow cabinet Horizontal laminar air flow cabinet
Room air enters the system from above the
HEPA filter; 99.99% particle-free air is
forced downward toward the work surface.
Room air enters the system from behind the
HEPA filter; 99.99% particle-free air is
forced in a back-to-front direction across the
work surface.

9. There are three types of biological safety cabinets:
Class I: The Class I biological safety cabinet is an open-front negative pressure
cabinet. The exhaust air from the cabinet is filtered by a high-efficiency
particulate air (HEPA) filter. The Class I biosafety cabinet will provide personnel
and environmental protection, but not product protection as dirty room air
constantly enters the cabinet front to flow across the work surface. Suitable for
work involving low to moderate risk agents where there is a need for containment
but not product protection. Hepa filter protect the env. By filtering air before it is
exhausted. Personnel protection by constant movement of air into the cabinet and
away from the user.
Class II: The Class II vertical laminar-flow biological cabinet is an open-front,
negative pressure vertical cabinet. hEpa filtered, vertical, unidirectional airflow
within the work area. Hepa filtered exhaust air to the room or exhaust to a facility
exhaust system. The exhaust air from the cabinet is also filtered by HEPA filters.
Thus, the Class II biosafety cabinet will provide personnel, environment and
product protection. While HEPA filters are effective for trapping particulates and
infectious agents, these filters will not capture volatile chemicals or gases .

10. Class III: The Class III cabinet is a totally enclosed ventilated cabinet of gas-
tight construction. Operations within the Class III cabinet are conducted
through attached rubber gloves. When in use, the Class III cabinet is
maintained through negative air pressure of at least 0.5 inches water gauge.
Supply air is drawn into the cabinet through HEPA filters. The cabinet exhaust
air is filtered by two HEPA filters, installed in series, before discharge outside of
the facility. The exhaust fan for the Class III cabinet is generally separate from
the exhaust fans of the facility's ventilation system.

11. BUILDING DESIGN ISSUES
Because the handling and storage of hazardous materials inherently carries a
high risk of exposure and injury, segregate laboratory and non-laboratory
activities to the extent possible.
1. Laboratory should be isolated and separated from food production areas,
preferable housed in a separate building
2. The fabric of the building must be ‘biosecure’ (no holes and cracks)
3. Large enough to accommodate all equipment and staff
4. Drainage from the lab. Should not flow through food production areas
and should enter the main flow downstream of the factory drains
5. Emergency exits at suitable points
6. Windows should be designed that cannot be opened, and if ledges are
present, they should be sloping to prevent them from being used as
storage areas
7. If windows require shading, externally fitted blinds or films should be
used.

12. Internal structure, fittings and
services
 Hot and cold water supplies and electric power points must be provided
 Gas is required for bunsen burners
 Dedicated hand washing facilities with hot and cold water supplies and supplied
with bactericidal soap and paper towel
 Dedicated double sink and drainer with hot and cold supplies and appropriate
cleaning chemicals for equipment washing
 Suitable steam extraction and ventilation systems for autoclaves, hot air ovens. Any
extraction system provided must safely exit to the external environment and not to
the manufacturing areas
 A water treatment system. Eg. Distillation unit, reverse osmosis
 The laboratory should be temperature controlled to facilitate incubator control and
provide a pleasant working environment
 All work surfaces (e.g., bench tops, counters, etc.) should be impervious to the
chemicals and materials used in the laboratory.
 The laboratory shall be designed so that it can be easily cleaned. Bench tops should
be of a seamless one-piece design to prevent contamination.
 Design laboratory workstations to accommodate the range of body dimensions that
may be using the workstations. For example, computer and microscopes
workstations may require height-adjustable work surfaces and chairs.

13. Work flow
A. Plan for microbiology laboratory in which no specific bacterial pathogen work is underta
(The hygienic integrity of lab. Should not be breached by staff , eg. From area 1, through are
7 to area 4, or from area 1 through areas 5 and 8 to areas 9 or 10
E
Entrance lobby
1
Cloak
Room
2
3
Laboratory
Coat change/
handwash
4
Sample booking in
7
Sample store
9
Main laboratory
6
Office
5
General store
8
Media preparation
10
Wash up/decontamination

14. Work flow
B. Plan for microbiology laboratory that includes a bacterial pathogen handling facility
(The hygienic integrity of lab. Should not be breached by staff , eg. From area 1, through are
7 to area 4, or from area 1 through areas 5 and 8 to areas 9 or 10
E
Entrance lobby
1
Cloak
Room
2
3
Laboratory
Coat change/
handwash
4
Sample booking in
7
Sample store
11
Main laboratory
6
Office
5
General store
8
Media preparation
10
Wash up/decontamination
9
Coat change/
handwash
Pathogen
laboratory
12
Hatch/
undercounter

15. EQUIPMENTS USED IN THE
LABORATORY
Autoclave Incubator Hot air oven
Inoculating
loop
Vortex mixer
/ shaker
Water bath
Heating
mantle
Hot plate
with
magnetic
stirrer
UV chamber
Inoculation
chamber
pH meter
Colony
counter
Microscope Refrigerator
Bunsen
burner
Spirit lamp
Micrometer
(stage and
ocular)
Balance
(Digital and
4-beam)
Thermometer
Membrane
filter set

16. LABORATORY OPERATION AND PRACTIC

17. LABORATORY
PERSONNEL &
STAFF
 Microbiological testing should be performed
and supervised by an experienced person, qualified
in microbiology or equivalent.
 Staff should have basic training in
microbiology and relevant practical experience
before being allowed to perform work.
 The laboratory should also maintain records of
all technical personnel, describing their
qualifications, training and experience.
 Personnel should be trained in necessary
procedures for containment of microorganisms
within the laboratory facility.
 Personnel should be trained in safe handling of
microorganisms.

18. Laboratory housekeeping
1. The laboratory must be kept tidy at all time.
2. Boxes of disposables etc. should be stored off the floor, and levels o
stocks held in the laboratory should be kept minimum. Bulk stocks
should be held in a suitable dedicated microbiology store.
3. A good disinfectant (for example hypochlorite based) must always b
available. Benches should be disinfected at the end of each working
period and as necessary to maintain a safe working environment.
4. A regular laboratory cleaning and disinfection programme must be
maintained to prevent floors, walls, ceilings, fitments and equipme
such as water baths and interiors of incubators and refrigerators fro
becoming source of infection for personnel, samples and media.
5. Soiled laboratory coats should preferably be autoclaved before bein
sent for laundering.

19. Laboratory waste disposal
1. Safe waste disposal systems must be provided, understood and use
efficiently by all staff. Waste must never be taken from the laborato
through food manufacturing areas.
2. Used pipettes, tips for pipettes and microscope slides should be
discarded into containers of disinfectant until they can be autoclav
prior to disposal.
3. Samples entering the laboratory for examination must be disposed
of through the laboratory and never returned to the factory. Unuse
food samples that have entered the laboratory should not be
consumed. Samples in the laboratory must be kept secure to preven
unauthorised removal.
4. Autoclave processes for decontaminating waste should normally
achieve a minimum of 121°C for atleast 30 minutes and preferably
45 minutes, or an equivalent heat process.
THE RESPONSIBILITY FOR SAFE DISPOSAL LIES WITH THE
WORKER RESPONSIBLE (THIS MEANS YOU !!!!)

20. LABORATORY SAFETYWaste materials awaiting disposal should be safely
stored. Flammable & halogenated solvents & other
contaminated waste liquids should not be poured into the
drains carrying the factory effluent. They should be
collected separately & disposed off suitably . There
should be separate waste containers for broken glass.

21. ENVIRONMENTAL MONITORING
 Bench surfaces used for sample and culture processing, hand was
basins, taps, handles and door seals, overhead structures, laminar
air flow cabinets, balances, laboratory blenders used be monitored
for their hygienic status:
 Visual assessment
 Swabs
 Contact plates
 Settle plates
 Records must be kept of the results

22. DOCUMENTATIONThe aim of documentation is :-
 To define specifications for all materials, method of
manufacture & control.
 To inform all concerned manufacturers, personnel
how when & why a batch is rejected.
 To provide audit trail for to permit investigation of
any suspected defective batch.

23. STANDARD
OPERATING
PROCEDURES
SOPs are basically required for :-
• Analysis of drugs
• Sample handling & accountability
• Receipt, identification, storage &
sampling of test & control articles.
• Cleaning, maintenance and calibration
of equipments
• Responsibilities of audit team
personnel.
• Healthy & safety precaution
• Storage & maintenance of microbial
cultures.
• Maintenance of animal rooms.
• Use & storage of reference standards
• Training programs & their frequency
• House Keeping
• Document control
• Retention & disposal of control
samples
A SOP is a procedure that has been
Determined, usually by a senior laboratory
staff, as the most appropriate means for
Carrying out a specified activity in the lab.

24. Fundamental requirements for achieving good aseptic and gen
microbiological practices:
1. All containers, tools, media and other test equipment/ materials used for directly handlin
test samples must be sterile.
2. Spreading and streaking techniques used for dispersing an inoculum over the surface of a
agar in a Petri dish (plate) must be undertaken with care to avoid contact with the edge of
plate which can lead to microbial growth that is difficult to assess and can lead to inaccura
results.
3. Mixing procedures for each inoculum with an agar, in the pour plate technique must be
undertaken with care to avoid splashing the sides and lid of the plates.
4. When a water bath is used for tempering bottles of molten agar, the bottles must be prope
dried using disposable paper towels prior to pouring the agar into Petri dishes. This preve
un-sterile water from the water bath from contaminating the inoculum, which would
otherwise add microorganisms to the test sample under examination and lead to inaccura
results.

25. Steps in sample processing procedure at which specific
aseptic practice is required
Process steps Aseptic handling specifically required
(YES)
Good microbiological practice
required (GMP)
Receipt of factory sample and storage of
sample
GMP
Weighing of test sample Yes
Addition of diluent Yes
Blending/homogenization GMP
Further dilution(s) Yes
Inoculation (media/petri dish) Yes
Addition of molten media Yes
Mixing/dispersion Yes
Incubation GMP
Assessment of growth GMP
Sub-culture for confirmation Yes
Incubation GMP

26. Test sample preparation and handling
1. All factory samples taken for microbiological tests should be examined within a timescale
appropriate to the sample, so that changes in the microbial composition of the sample
between sample collection and testing are minimised.
(a) A chilled product taken from a manufacturing line should be examined ideally within 1
of sample collection to ensure the sample’s condition adequately reflects the
manufacturing circumstances.
(b) All samples must be packed cleanly for transport taking care to keep raw or ‘dirty’ samp
e.g. dusty and environmental samples well separated from clean samples.
(c) When microbiological testing is sub-contracted to an external laboratory, ‘same day tes
should be achieved for all factory samples held in refrigerated storage.
(d) A specified quantity of test sample e.g. 10 g or 25 g is removed from the factory sample
using sterile implements and good aseptic practices.
2. (a) For solid food materials, test sample treatment must ensure good dispersion of the sam
in the diluent, e.g. using a laboratory blender with a min. blending time of 30 seconds,
max. of 1 minute.
(b) Heat (35-40°C) is used to aid dispersion, e.g. for high fat (>30%) samples; heat should b
used with caution where samples are to examined for psychrotrophic bacteia

27. Contd..
3. Care is required in test sample treatment to ensure that there is minimum shock to organism
and maximum recovery by using, for example,
 Resuscitation techniques where necessary, e.g. dried products should be rehydrated
gently using a method appropriate for the sample type
 Extra dilution, quenching agents or special diluents to inactivate inhibitory substan
such as sanitizers or essential oils naturally present in foods such as onion, garlic and
some herbs and spices, as well as some components of cocoa materials alone.
4. Diluted test sample must be inoculated into growth media within 20 minutes of their initia
dilution and, preferably, should be re-mixed immediately before being used to inoculate m
5. Where diluents for primary dilution are pre-prepared in specific volumes, e.g. 90 ml and 18
, care must be taken to ensure the accuracy of these volumes.
6. A laboratory should have suitable control systems to ensure that contaminated diluents or
media will be recognized e.g. Sterility checks of incubated uninoculated plates.
7. Surface plating media and broth media must not be used directly from the refrigerator. Med
should be allowed to equilibrate to room temperature before use.
8. As a general rule, all prepared ‘pour plate’, ‘spread plate’ and ‘streak plate’ petri dishes shoul
be incubated in an inverted position to prevent condensation from wetting the agar surface
causing colonies to merge. However, the main exception to this are the plates used to grow m
colonies, where mould spores may be dislodged by turning the plates over and over.

28. 9. Efficacy check of prepared media:
Positive control: particularly useful for selective-diagnostic media to ensure the ‘target’
organisms, e.g. Salmonella, can grow well and produce the relevant diagnostic colony
characteristics.
Negative control: These, again, are particularly useful for selective diagnostic media to
ensure that competitor organisms are either inhibited or produce colonies that are
distinguishable from the target organism. For e.g. E. coli on selective culture media for
Salmonella.

29. Sampling and sample terminology
Factory sample
Laboratory
Weigh test sample, e.g. 10g, 25 g
For detection tests
Add pre-enrichment/
Enrichment medium
Blend to produce test sample suspension
Incubate
Assess growth Confirmatory test as required
For enumeration tests
Add primary diluent
Blend to produce test sample suspension
Inoculation of medium
Incubate
Assess growth

30. LABORATORYACCREDITATION
 Accreditation is an approved procedure by which
regulatory authorities or authorised body accord formal
recognition to a laboratory to undertake specific task,
provided that the pre-defined standards are met by.
 It involves re-assessment and surveillance at regular
intervals so as to ensure that commitment to quality is a
continuous process.

31.  The organisation providing accreditation to the laboratories in
India is the National Board Accreditation for Testing and
Calibration Laboratories (NABL), Department of Science
and Technology, Government Of India.
 The process of accreditation takes into account all the
parameters as per ISO 15189, which is an International
Standard.
 The Technical parameters of the standard include
requirements of manpower and their competence,
equipment, environment, material and use of standard
laboratory methods.

32.  In these schemes, an annual inspection of the laboratory's quality systems
procedures and practices is carried out and reported by independent inspector
 In addition to an annual audit by an independent body, laboratories also particip
in one or more External Quality Assessment (EQA) schemes.
These tests provide a challenge to the whole laboratory operation and
procedures including:
 Sample receipt
 Media preparation and quality control
 Sample handling procedures and traceability
 Staff practices
 Method efficacy
 Subscription by the laboratory to these schemes can provide a useful method f
helping to demonstrate the credible operation of the laboratory.