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Inoculation Techniques in Microbiology

Challenge Testing

Method · November 2014

DOI: 10.13140/RG.2.2.17433.93286

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Grzegorz Rachon
Campden BRI
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 Inoculation techniques in Microbiology Challenge Testing
Grzegorz Rachon MSc

Inoculation validation
The main dilemma when performing microbiology challenge testing, is the selection and validation of a method
of inoculation. The bacterial strains selected for challenge testing should always be associated with the product
being tested, ideally by being natural isolates, e.g. from earlier outbreaks or strains isolated from similar
products, similarly processed. Preparation of the bacterial inoculum is also very important; strains should be
prepared in carefully selected conditions so stress on entering the new environment, is minimized. The method
of inoculation must always be validated and must not change the product structure or chemical or physical
characteristics, e.g. water activity, pH, gas within the packaging. etc. To achieve a satisfactory inoculation, many
methods have been developed and are in general use: direct inoculation, using micro-pipettes are generally
used for liquid samples where bacteria are dispersed in the tested product: spraying techniques, dispersing
bacteria on the surface of the product can be used: MAP or vacuum-packed products, are usually inoculated
using syringe and needle injecting through inoculation tape or inoculating opened packs and re-packing after
inoculation: products of low water activity, where addition of liquid phase is not acceptable, are inoculated using
glass beads covered with biofilm, or silicon sand, chalk or product dust may be used as bacteria carriers; these
latter methods are often used for inoculation of powders, seeds and nut kernels, transferring bacteria into the
product or dispersing them on the product surface. The number and distribution of the inoculated cells must be
determined before proceeding with the challenge test.

Level of Inoculation
The level of inoculation is also very important; too high a number of bacteria added to the product can interfere with
the natural microflora of the tested product and therefore cannot be used, and too low initial levels may not survive
an adaptation phase after inoculation. Thus, the number of viable cells in the washed and ready-to-use microbial
cocktail is critical and must be obtained prior to inoculation. Since cell cocktails used for challenge testing should
always be made on the day of inoculation, enumeration using alternative rapid methods, is essential. Only one
exception from this rule is acceptable that refers to bacterial spores which are quite stable when stored in chilled
conditions and can be prepared and enumerated several days before inoculation, using standard viable count
techniques. Microscopic rapid enumerations of yeast or mould spore cocktails can be performed using counting
chambers (Fuchs-Rosenthal chamber) and is widely used and always gives satisfactory results compared with
results obtained from viable count techniques; only one limitation of this method is size of microorganisms as small
cells can be easily missed during counting and contribute to large reading errors. For small bacteria (Salmonella,
E.coli, Listeria, Lactobacillus, etc.) rapid enumeration of cells using optical density measurement (OD) has been
introduced and is used in Leatherhead on a daily basis. In this method the Optical Density of a cocktail and its
dilutions is measured and the number of cells is calculated from earlier prepared concentration curves of OD vs.
Cell Concentration (cfu/mL).

Optical density of bacterial suspensions at 350 nm

2.00E+09

1.90E+09

1.80E+09

1.70E+09 y = 3E+08x3 + 4E+08x2 + 1E+09x + 1E+07

R² = 0.9998
1.60E+09

1.50E+09

1.40E+09
y = 1E+09x4 - 2E+09x3 + 2E+09x2 + 6E+08x + 3E+07
1.30E+09 R² = 0.9997

1.20E+09

1.10E+09
Salmonella
cfu/g

1.00E+09 E.coli
9.00E+08
Staph
y = 3E+08x2 + 5E+08x - 3E+06
R² = 0.9997 Cocci
8.00E+08
Lactobacilli
7.00E+08

6.00E+08
y = 3E+08x3 - 1E+08x2 + 5E+08x + 282875
5.00E+08 R² = 0.9995

4.00E+08

3.00E+08

2.00E+08
y = 2E+08x2 + 4E+07x + 2E+07
R² = 0.9517
1.00E+08

0.00E+00
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20

OD

If Optical Density is read, the number of cells can be read either directly from the graph or by
simple calculation from the curve equation. For example, the equation for E.coli y = 1E+09x4 -
2E+09x3 + 2E+09x2 + 6E+08x + 3E+07 can be transformed to Excel formula:
y=1E+09*POWER(x,4)2E+9*POWER(x,3)+2E+09*POWER(x,2)+6E+08*x+3E+07 and the
number of cells will be automatically calculated.

For further information please contact Grzegorz Rachon, Email:

grzechoo2@hotmail.com
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