Sample Preparation, Dilution and Plating

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MICROBIOLOGICAL MEDIA & SAMPLE PREPARATION, PRIMARY DILUTION, FURTHER

DILUTION & OTHER DILUTION
1.0 RESPONSIBLE PERSON/S
QC Analyst for Microbiology

2.0 PERSONAL PROTECTIVE EQUIPMENT (PPE)
 Laboratory gown
 Safety laboratory glasses or goggles
 Head cap
 Surgical Mask or N95
 Disposable non-powdered latex gloves
 Closed-toed white shoes
3.0 MATERIALS/TOOLS/EQUIPMENTS
 Peptone
 Distilled H2O
 Dilution/Corning Bottles
 Beakers, Volumetric flasks, Graduated cylinders
 Autoclave Steam Indicator Tape
 Sterilizer/ Autoclave
 Analytical balance
 Tap load balance
 Sterile bags
 pH Meter
 Spatulas
 Magnetic stirrer
 Stirrer/stirring rod
 Autoclavable biohazard bag
4.0 DILUENT PREPARATION
4.1 0.1% PEPTONE DILUENT
a. Weigh 1g peptone and dilute to 1L distilled water to prepare 0.1% peptone diluent.
Note: Do not use buffers containing citrate, bisulfite, or thiosulfate; they can inhibit
growth.
b. Stir the diluent until peptone powder homogenizes. Get the pH of the prepared 0.1%
Peptone diluent.
c. Adjust pH of the diluted sample between 6.5 and 7.5.
 For acid products, use 1 N NaOH
Preparation of 1N NaOH
Dissolve 4g NaOH in 1L deionized or distilled water
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 For alkaline products, use 1 N HCI
Preparation of 1N HCl
Dissolve 3.65g HCl in 1L deionized or distilled water
d. Transfer 0.1% peptone diluent to a dilution bottle (i.e. 90ml of peptone diluent to perform
a 1:10 dilution).
e. Proceed to sterilization.
5.0 STERILIZATION OF MEDIA AND OTHER TOOLS USING AUTOCLAVE
a. Placed all prepared media, pipette tips, forceps, empty corning bottles in an
autoclave.
b. Attached a sterility tape on them prior to sterilization.

Note: Autoclave must be set at 121°C (249°F) at 15psi for 15mins.
c. After approximately 2 hours (for heating up and cool down process of autoclaving),
check for occurrence of color change on sterility tape for validation of complete
sterilization.
d. Store all sterilized prepared media in a refrigerator with a temperature set at 2-8°C
and the other tools in a sealed pouch bag at room temperature.
6.0 Aseptic technique
When handling samples, aseptic technique is essential to avoid

contamination of the sample and to protect the laboratory worker from infection.
The following points must be observed when preparing samples.
a. Caps and lids from containers should not be placed on the workbench but
retained in the hand whilst the sample is being processed. Caps and lids
must be replaced as soon as possible.
b. Keep samples away from the face when opening culture containers.
c. Minimize the production of aerosols by opening caps slowly, after mixing

allow universal to stand for a minute prior to opening.
d. If forceps or spatula are used when handling samples these must be

sterilized by autoclaving or decontaminated using 70% ethanol prior to use.
Safety eyewear and gloves must be worn.
e. All consumable items used to prepare samples such as, loops, pipettes,
pipette tips, hockey sticks, bags and containers must be sterile single use
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items.
f. When pouring liquid media ensure that the mouth of the bottle does not
come into contact with samples or non-sterile items. If this occurs, discard
the media.
g. Samples should be prepared in such a way as to ensure that no bacterial

contamination is introduced by contact with hands or contact with unsterile
surfaces or items.
7.0 Sample preparation
Homogeneous samples including powders like flour and free flowing liquids and
concentrates should be mixed well before removing a portion for testing (for
example, by shaking 25 times). Do not shake powders immediately before testing
as the environment may become contaminated by dust particles.
Packaged products should be opened aseptically, and if necessary and wearing

safety glasses and gloves (if the package cannot be opened without risk of
external contamination), the external surface of the package should be
disinfected by wiping with 70% alcohol or aseptic wipes to avoid
contamination when opening.
The temperature of the diluent should be approximately the same as that of the
test sample.
Use aseptic techniques throughout all sampling and handling procedures to

avoid the introduction of extraneous microorganisms. Containers, stomacher
bags and any utensils used for sampling must be sterile.
8.0 Test portion and initial suspension (primary dilution)
a. Weigh or measure the test portion, to a tolerance of ±5 %, into a sterile

container or plastic bag.
b. A mass of m g or a volume of V ml (minimum 10 g or 10 ml, unless

otherwise stated) representative of the laboratory sample shall be used.
NOTE Using larger test portions than the minimum specified above will increase
the reliability of enumeration test results.
c. Add a quantity of diluent equal to 9 × m g or 9 × V ml to prepare a

primary decimal dilution. This quantity shall be measured, preferably by
mass, to a tolerance of ±2 %, but measurement by volume is also permitted
provided that the tolerance is no more than ±2 %. Other primary dilutions
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of lower or higher ratios of diluent to test portion may be required for

special purposes; these are prepared using the same tolerances.
d. Certain types of product result in viscous or thick initial suspensions

when prepared with the usual 1 in 10 dilution and additional diluent may be
necessary to facilitate further testing. In such cases, the diluent shall be
added in other ratios (e.g. 1 in 20, 1 in 50, 1 in 100) until a satisfactory initial
suspension for further operations is achieved. These non-standard ratios
shall be taken into account in subsequent operations, particularly in the
calculation and expression of results.
e. In other cases where low numbers of microorganisms are sought and

the product being tested will produce a suitably liquid initial suspension, it
may be desirable to prepare the suspension at lower ratios (e.g. 1 in 2 or 1
in 5). However, use of such lower ratio initial suspensions may result in
imbalance of the inoculum to medium ratio in subsequent selective plating
in or on solid media or into liquid media (e.g. inhibition of microbial growth
by the increased concentration of food components) and this approach
shall be used with caution and verified on a case-by-case basis.
f. To avoid damage to microorganisms by sudden changes in temperature,

the temperature of all diluents shall be approximately the same as the
laboratory ambient temperature, except where otherwise specified for
particular products.
g. Homogenize the mixture. Allow large particles to settle, if necessary, for

up to 15 min. Filtration systems giving equivalent results, such as plastic
bags with integral filter liners, may also be used.
h. For enumeration of spores, heat treatment of the initial suspension (e.g.

10 min ± 1 min at 80 °C ± 5 °C) shall be performed immediately after
preparation of the suspension, followed by rapid cooling (e.g. under
running cold water) to minimize subsequent loss of the target spores.
9.0 Further dilutions
9.1 Decimal dilution series
a. For a decimal dilution series for use in enumeration tests, transfer,

using a pipette (6.5), 1 ml ± 0,02 ml of the initial suspension into a
tube containing 9 ml ± 0,2 ml of sterile diluent at laboratory ambient
temperature (unless otherwise stated in the specific standard).
Avoid any contact between the pipette containing the inoculum and
the sterile diluent to minimize cross-contamination potential.
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NOTE If a greater volume is needed to perform a large number of tests or

replicate tests, a determined volume (more than 1 ml) of the initial suspension,
with a tolerance of ±2 %, can be added to a tube containing a nine-fold volume of
sterile diluent.
b. For optimal precision, do not introduce the pipette more than 1 cm

into the initial suspension and avoid withdrawing particles of the
food product in the inoculum.
c. Mix thoroughly, preferably by using a mechanical stirrer (6.3) for

5s to 10 s, to obtain a 10−2 dilution.
d. If necessary, repeat these steps using the 10−2 and subsequent

dilutions and a new sterile pipette or tip for each operation, to obtain
sufficient (10−3, 10−4, etc.) dilutions to enumerate the appropriate
number of microorganisms at the optimum range for counting. The
sequence of operations for preparing multiple decimal dilutions
suitable for different anticipated levels of contamination and using
the pour plate technique is shown in Figure 1.
Figure 1 — Sequence for preparation of multiple decimal dilutions — Example for

pour plate technique
9.2 Other dilution series

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a. Prepare any other dilution series required for special purposes,

e.g. 1 in 2 (1 ml to 1 ml), 1 in 5 (1 ml to 4 ml), in an identical way by
using different ratios of the initial suspension to diluent. Record the
ratio and take it into account in subsequent steps, such as the
calculation and expression of results.
10.0 Duration of the procedure
a. The time between the end of the preparation of the initial suspension
and the moment when the inoculum comes into contact with the final
culture medium shall not exceed 45 min.
b. The time between the preparation of the initial suspension and the
beginning of preparation of any subsequent dilutions shall not exceed
30min.
c. If the ambient temperature of the laboratory is high and outside the

recommended range (>27 °C), these two maximum durations should
be reduced to minimize potential for microbial growth and consequently
higher results.
d. If a resuscitation period to maximize recovery of damaged

microorganisms is required by the specific International Standard, this
shall be timed once the initial suspension has been prepared and the
subsequent dilution steps commenced immediately after this period has
ended.
References
1. BS EN ISO 6887–1:1999. Microbiology of food and animal feeding stuffs –

Preparation of test samples, initial suspension and decimal dilutions for
microbiological examination. Part 1 : General rules for the preparation of the initial
suspension and decimal dilutions.
2. BS EN ISO 6887-2:2003. Microbiology of food and animal feeding stuffs –

microbiological examination. Part 2 : Specific rules for the preparation of meat and
meat products.

microbiological examination. Part 3 : Specific rules for the preparation of fish and
fishery products.

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microbiological examination. Part 4 : Specific rules for the preparation of products

other than milk and milk products, meat and meat products, and fish and fishery
products.

microbiological examination. Part 5 : Specific rules for the preparation of milk and
milk products.
6. BS EN ISO 6887–1:2017. Microbiology of the food chain – Preparation of test samples,

initial suspension and decimal dilutions for microbiological examination. Part 1 : General
rules for the preparation of the initial suspension and decimal dilutions.
7. Health and Safety Executive. Biological Agents: Managing the risks in laboratories
and healthcare premises; 2005. http://www.hsegov.uk/biosafety/biologagents.pdf
8. Control of Substances Hazardous to Health Regulations 2002. General COSHH.

Approved Code of Practice and Guidance, L5. Suffolk: HSE Books; 2002.
9. Health and Safety Executive. Five steps to risk assessment a web version of
leaflet INDG163(rev3), revised 06/11
10. ISO 7218: 2007 incorporating Amd 1:2013. Microbiology of food and animal
feeding stuffs – General requirements and guidance for microbiological
examinations.
11. Public Health England (2013) Sample processing and result entry in STARLIMS
Microbiology Services. Food, Water & Environmental Microbiology Standard
Method FNES6 (Q12) Version 3.

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MICROBIOLOGICAL MEDIA & SAMPLE PREPARATION, PRIMARY DILUTION, FURTHER

1.0 RESPONSIBLE PERSON/S

QC Analyst for Microbiology

4.0 DILUENT PREPARATION

4.1 0.1% PEPTONE DILUENT

c. Adjust pH of the diluted sample between 6.5 and 7.5.

 For acid products, use 1 N NaOH

 For alkaline products, use 1 N HCI

5.0 STERILIZATION OF MEDIA AND OTHER TOOLS USING AUTOCLAVE

b. Attached a sterility tape on them prior to sterilization.

6.0 Aseptic technique

When handling samples, aseptic technique is essential to avoid

The following points must be observed when preparing samples.

c. Minimize the production of aerosols by opening caps slowly, after mixing

d. If forceps or spatula are used when handling samples these must be

g. Samples should be prepared in such a way as to ensure that no bacterial

7.0 Sample preparation

Packaged products should be opened aseptically, and if necessary and wearing

Use aseptic techniques throughout all sampling and handling procedures to

8.0 Test portion and initial suspension (primary dilution)

a. Weigh or measure the test portion, to a tolerance of ±5 %, into a sterile

b. A mass of m g or a volume of V ml (minimum 10 g or 10 ml, unless

c. Add a quantity of diluent equal to 9 × m g or 9 × V ml to prepare a

of lower or higher ratios of diluent to test portion may be required for

d. Certain types of product result in viscous or thick initial suspensions

e. In other cases where low numbers of microorganisms are sought and

f. To avoid damage to microorganisms by sudden changes in temperature,

g. Homogenize the mixture. Allow large particles to settle, if necessary, for

h. For enumeration of spores, heat treatment of the initial suspension (e.g.

9.0 Further dilutions

9.1 Decimal dilution series

a. For a decimal dilution series for use in enumeration tests, transfer,

NOTE If a greater volume is needed to perform a large number of tests or

b. For optimal precision, do not introduce the pipette more than 1 cm

c. Mix thoroughly, preferably by using a mechanical stirrer (6.3) for

d. If necessary, repeat these steps using the 10−2 and subsequent

Figure 1 — Sequence for preparation of multiple decimal dilutions — Example for

9.2 Other dilution series

a. Prepare any other dilution series required for special purposes,

10.0 Duration of the procedure

c. If the ambient temperature of the laboratory is high and outside the

d. If a resuscitation period to maximize recovery of damaged

1. BS EN ISO 6887–1:1999. Microbiology of food and animal feeding stuffs –

2. BS EN ISO 6887-2:2003. Microbiology of food and animal feeding stuffs –

3. BS EN ISO 6887-3:2003. Microbiology of food and animal feeding stuffs –

4. BS EN ISO 6887-4:2003. Microbiology of food and animal feeding stuffs –

microbiological examination. Part 4 : Specific rules for the preparation of products

5. BS EN ISO 6887-5:2010. Microbiology of food and animal feeding stuffs –

6. BS EN ISO 6887–1:2017. Microbiology of the food chain – Preparation of test samples,

8. Control of Substances Hazardous to Health Regulations 2002. General COSHH.

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