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Indian Journal of Medical Microbiology, (2005) 23 (3):159-163

Special Article

QUALITY CONTROL OF CULTURE MEDIA IN A MICROBIOLOGY

LABORATORY
*S Basu, A Pal, PK Desai

Abstract
The nature of reporting of a microbiology laboratory depends upon the quality of the culture media used. Quality of
media directly affects the observations and inferences drawn from the cultural characteristics of microorganisms. Checking
of different parameters of media such as growth supporting characteristics, physical characteristics, gel strength and batch
contamination can help to assess their quality. There are different methods to check all these parameters systematically.
The meticulous performance of quality control of culture media can assure precision in reporting.

Key words: Quality control, culture media

Culture media play a pivotal role in any microbiology
laboratory. They are widely employed for isolation,
identification and sensitivity testing of different pathogenic
microorganisms. Most of the laboratories usually prepare their
own media for routine diagnostics as well as research
purposes. However, to ensure that the media is of good quality
and capable of giving satisfactory results, proper quality
management system is essential. For that purpose certain
parameters of media prepared should be thoroughly checked
and then passed for laboratory use.
This article is aimed to provide certain information about
the parameters to be considered and the tests to be performed
to ensure proper quality of media. We have divided quality
control procedures of media in different sections according
to the parameters that need evaluation.
Raw material parameters
The quality of the media depends directly upon the quality
of the raw materials used for their preparation. Water is the
most important raw material used for the preparation of
culture media. The parameters to be checked are presence of
copper ions, conductivity and pH. Ideally there should be no
copper ions present in water because it is inhibitory for the
growth of microorganisms. The conductivity should be less
than 15 µS (microsiemens). The pH of the water should be
slightly on the acidic side, but should not be less than 5.5. 1
The quality of petri dishes used for pouring of media is
also an important factor. Normally petri dishes are ethylene
oxide (EtO) sterilized or gamma irradiated. If EtO sterilized
they should be then checked for residual EtO toxicity, as this
may affect the growth of the microorganisms. The maximum
*Corresponding author (email: <somansu@rediffmail.com>)
R&D Department, Microbiology Division, Span Diagnostics Ltd.,
Udhna, Surat – 394 210, Gujarat, India.
Received: 21-06-2004
Accepted: 19-10-2004
permissible limit for residual EtO is 1 µg/g and it can be
measured by standard gas chromatographic methods.2 Only
borosilicate glassware should be used because soda glass can
leach alkali into the media.
There are various additives used in preparation of media.
Blood is the most important one of them. Hence the quality
of the blood plays an important role in the performance of
the blood containing media. The sterility, homogeneity,
viscosity and colour of the blood should be scrupulously
checked before it is used for media preparation. For other
additives the certificate of analysis and sterility conditions
should be considered.
Sterilization parameter
Sterilization of the media plays an important role in the
quality of the media. Generally autoclaving is carried out for
sterilizing the media. However, the time of autoclaving and
the quantity of media sterilized should be closely regulated.1
Heat treatment of complex culture media may result in its
nutrient destruction either by direct thermal degradation or by
reactions between the components. Therefore, it is very
important to optimize the heating process to minimize heating
damages. The suggested cycle is stage 1:20-121°C, stage 2:
<100-121°C, stage 3:121-121°C and stage 4:121-80°C.
The volume of the media in one sterilization batch should
be kept small, ideally two litres. Regular checking of the
sterilization process by indicators should be done; temperature
and pressure should also be constantly monitored. Sterilization
indicators such as biological indicators and Bowie Dick test
are available to check the efficiency of the process. Biological
indicators such as spores of Bacillus stearothermophilus3 can
be used to check the spore killing efficacy.
Physical parameters
The gross physical appearance of media often suggests the
quality. Media prepared should be screened for physical

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160 Indian Journal of Medical Microbiology
characteristics4 such as excessive bubbles or pits, unequal
filling of plates (uniform leveling), cracked medium in plate
and freezing or crystallization.
All the above mentioned characters can be checked
visually by naked eye. However, for unequal filling of plates,
thickness of medium can be checked at four points. These four
points are the two ends of the two diameters of the plate,
which are at right angles to each other. Thus all the four sides
can be simultaneously checked. The thickness at the four
points is noted down and the mean thickness is determined
and reported as mean thickness of the medium in the plate,
which must be 4.0 ± 0.2 mm.5
The pH value of the medium is also one of the important
physical characters, which must be checked. 1 It can be
measured while preparation of the medium before and after
autoclaving by using the standard pH meter after proper
calibration with standard buffers.
Microbiological parameters
Growth supporting characteristics is the most important
parameter while conducting quality control of media. Standard
inoculating procedures should be used. The results should be
examined both qualitatively and quantitatively and while
testing new lots, both previous batch and new batch should
be simultaneously grown.1
National committee for clinical laboratory standards
(NCCLS) has laid down certain guidelines for the control
organisms to be used for every medium, the desired inoculum
concentration and their expected growth results. Inoculum for
every medium can be prepared according to the following
method:
The control organism is inoculated in soyabean casein
digest (SCD) broth and incubated for 4 hours to get a cell
density comparable to 0.5 McFarland’s standards. The
standard suspension should give a colony count of 10 7- 108
cfu/mL (0.08-0.1 absorbance at 625nm). A 10 µL quantity of
inoculum of 1in 10 and 1 in 100 dilution in normal saline or
in SCD broth should be used for selective and nonselective
Table 1: Format for quality control record
Name of media prepared:
Date of preparation:
Conductivity of water:
pH of water:
Sterility status of blood / additives:
Sterilization details:
Media Control Organism NCCLS recommended
results
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160 CMYK
vol. 23, No. 3
media respectively. 6 These diluted inocula are used to
ascertain the growth supporting capacity of the media. The
inoculation is done in duplicates for each type of inoculum.
After inoculation, the plates are incubated at 37°C for 24 hours
and their growth and colony characteristics are observed. The
results can be reported by mentioning presence or absence of
growth and the growth characteristics in a tabular form as
shown in table 1.
In practice, the absolute measurements of growth of
microorganisms are either time consuming or require
sophisticated instruments. Colony size may be used to see the
performance but it is again an insensitive indicator. Colony
characteristics are subjective and very difficult to record.
Methods like ecometric method and proportional method,
which gives us comparative data, are therefore suitable for
routine quality control of microbiological performance of
culture media. They can be utilized to check both the growth
as well as inhibition characteristics of media.7
Ecometric method
This is a simple and numerical method. Both absolute
growth index (AGI) and relative growth index (RGI) can be
determined.8 The method is based on streaking of inoculum
to extinction. This method can be used to compare results with
previous batches of same medium or between selective and
nonselective media.
In this method five millilitre of brain heart infusion broth
is inoculated with loopful of chosen test organism and
incubated for four hours. The petri dish of given medium
should be divided into 4 quadrants and streaking lines should
be drawn on them as shown in figure 1.
One microlitre loop has to be charged with culture and
streaked in the way as A1, B1, C1, D1, A2, B2 …… upto D5
without flaming or recharging the loop. The procedure has to
be repeated for the control plate. After incubation the last point
should be noted in both test and control on which growth
occurs. These are the end points of test and control media.
These readings can be used to calculate the absolute growth
Results of Results Remarks
previous batch of new batch
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July 2005 Basu et al – Quality Control of Culture Media 161

Figure 2: Schematic diagram of productivity ratio

the dilution to be used as shown in figure 2.

tarting with highest dilution, each drop of the dilutions is

Figure 1: Schematic diagram of the ecometric method
placed on the relevant quadrant. Same step is repeated for
Table 2: Absolute growth index end points control plate. Each drop is spread in the corresponding
quadrant and incubated at 37°C for 18 hours. The colonies of
A1 = 5 B1 = 10 C1 = 15 D1 = 20 the lowest dilution are counted for both test and control plates.
A2 = 25 B2 = 30 C2 = 35 D2 = 40
A3 = 45 B3 = 50 C3 = 55 D3 = 60 Contamination parameter
A4 = 65 B4 = 70 C4 = 75 D4 = 80 This is a very crucial parameter for the determination of
A5 = 85 B5 = 90 C5 = 95 D5 = 100 the quality of media. The batch must be scrupulously checked
for contamination before passing for laboratory use. It is also
index (AGI) and relative growth index (RGI) of the medium. suggested that the whole batch of the prepared media be
The AGI is obtained by noting the end points (Table 2). checked for contamination by keeping the plates at least for
three days at room temperature.9 Alternatively, two plates from
The RGI is a comparison of the AGI of the test plate and
the test batch can be taken and placed into the incubator set
control plate.
at 37°C for 24 hours. After required incubation, the plates are
AGI Test checked for any growth. If there is any growth, the process
RGI = x 100 is repeated, taking again two plates from the same batch. If
AGI Control contamination occurs again, it is inferred that contamination
has occurred in the prepared batch. As per recommendations
The RGI varies according to the intended purpose of
more than 10% contamination requires the batch to be
medium. In order to check efficiency the RGI should be close
discarded.10
to 100%, whereas for checking inhibition it should be closer
to 0%. The performance of a selective agar can be tested using Gel strength parameter
an organism that has been selected to check isolation and
using another that has been selected to check inhibition. Gel strength is an indication of level of solidification of
the agar in the medium. The gel strength is measured by using
Productivity ratio a tripod stand with a central rod that is used to impart pressure
on the agar. The lower end of the rod has a spherical portion,
Determining the productivity ratio (PR) of a medium is which rests on the medium surface. The upper end of the rod
another method of determining the performance relative to the has a platform on which standard weights are placed. The
control medium, which should be a nutritious agar like spherical portion of the central rod is placed on the medium
tryptone soy agar (TSA). The inoculum used must be same and weights are placed on the upper platform one by one and
for both media and PR is calculated by counting the colonies observed for some time. The process is continued until the
on test and control media. agar breaks under the weight of the central rod. While
No. of colonies on test x dilution factor calculating the gel strength the weight of the central rod
PR = should be deducted. The force imparted by the rod on the agar
No. of colonies on control x dilution factor W
surface can be calculated by the formula: 2
πr
The method follows the modified Miles & Misra
technique. A tenfold dilution of an overnight culture of test Where, w = weight kept on the platform, r = radius of
organism is prepared in buffered peptone water. The test plates the spherical portion at the lower end of the central rod and
are divided into 4 quadrants and each quadrant is marked with π = 3.14.

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162 Indian Journal of Medical Microbiology
A gel strength of about 300 – 500 dynes/cm2 will give
satisfactory results (unpublished data)
Trouble shooting guide
Table 3 gives information about the various problems
found in culture media and their probable causes.
Present scenario
The quality control (QC) of media used in clinical
microbiology laboratory remains critical for accurate and
acceptable isolation of pathogens from infected patients.
Testing of media using standard protocol can save time and
resources.11 In a recent QC compliance study in Ontario, it
was found that NCCLS QA recommendation was not
followed at all. Moreover, recommended ATCC strains were
used only in half of the participating laboratories.12 Lot failure
rates for all media ranged from 0.10% to 9.87% (average
1.01%) 13. The reasons for failure were no growth (39.9%),
no inhibition (18.6%), non-sterile (17.9%), haemolysis (7.2%)
and surface defect (16.3%).
In another study it was found that the batch failure rates
Table 3: Trouble shooting guide for culture media
Soft Gel Excess heat, pH too low causing acid
hydrolysis, inaccurate weighing,
inadequate mixing, agar not dissolved,
poor quality of agar in dehydrated culture
media
pH incorrect Contaminated glassware, impure water,
overheating, chemical contamination, pH
taken at wrong temperature, pH
measuring equipment faulty or poorly
standardized, deterioration of dehydrated
medium.
Abnormal colour Impure water, dirty glassware,
deterioration of dehydrated medium,
excess heat, pH incorrect.
Darkening Excess heat, deterioration of dehydrated
medium.
Precipitation Excess heat, deterioration of dehydrated
medium, impure water or glassware.
Toxicity Excess heat (scorching or burning),
deterioration of dehydrated medium.
Poor growth Contaminated water or glassware,
deterioration of dehydrated medium,
incorrect weighing and mixing. Excess
heat.
Poor selective or Contaminated water or glassware,
incorrect weighing and mixing,
differential deterioration of dehydrated
medium. Excess heat properties
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162 CMYK
vol. 23, No. 3
were rare (0.5%).14 Krisher et al pointed out that 41 out of
109 respondents used only 50% or less information from
NCCLS M22-A2. 15 In the recent document of NCCLS M22-
A3, media are divided on the basis of the extrapolated quality
control failure rates.6 Media with extrapolated failure rate
more than 0.5% require complete quality control by the user
and are known as nonexempt media such as nutrient broth,
Todd-Hewitt broth, MacConkey sorbitol agar, chocolate agar
with IsoVitaleX,® Campylobacter blood agar, brain heart
infusion (BHI) both with 5% sheep blood / chloramphenicol
and cycloheximide. Those with failure rate less or equal to
0.5% are exempt media for which minimal quality control is
recommended. It will minimize the cost and unnecessary QC
duplication.
References
1. Bridson E.Y. Culture Media In: The Oxoid Manual 8th edn.
OXOID England: Limited Hampshire; 1998. p. 2-8.
2. Page B. ISO Standard redefines limits for ETO residuals. MDDI
archive 1996.
3. Perkins JJ. Sterilizer control, sterilization indicators and culture
tests. In: Principles and methods of sterilization in health sciences.
Illinois: Charles C. Thomas Publishers; 1969. p. 483-500.
4. Evans GL, Bell RH, Cunningham LV, Ferraro MJ, Maltese AE,
Pienta PA, et al. Quality Assurance for Commercially Prepared
Microbiological Culture Media-; Approved standards- 2nd edn.
NCCLS M22-A2,1996:16.
5. Ferraro MJ, Wikler MA, Craig WA, Dudley MN, Eliopoulus
GM, Hecht DW, et al. Performance Standards for Antimicrobial
Disk Susceptibility Tests; Approved Standards 8th edn. NCCLS
M2-A8,2003:23.
6. Krisher K, Callihan DR, Jones RN, Luper DC, Miller JM, Sharp
SE, et al. Quality Control for Commercially prepared
Microbiological Culture Media; Approved Standards-3rd edn.
NCCLS M22-A3,2004:24.
7. Anonymous. Quality Control of Culture Media. In: Culture Media
Manual LAB M. (IDG Ltd., United Kingdom) 2002. P. 16-8.
8. Mossel DAA, Trees MG, Laarhoven BV, Anniek MT,
Lightenberg M, Maria EBW. Quality assurance of selective
culture media for bacteria, moulds and yeast: An attempt to
standardization at international level. J Appl Bact 1983;54:313-
27.
9. Arora DR. Quality Assurance in Microbiology. Indian J Med
Microbiol 2004;22:81-6.
10. Duguid JP, Collee JG, Fraser AG, Aikman KW. Organization of
the clinical bacteriology laboratory; quality assurance In: Mackie
& McCartney Practical Medical Microbiology 14 th edn.
Churchill Livingstone, London;1996. p. 1-16.
11. Weenk GH, vd Brink J, Meeuwissen J, van Oudenallen A, van
Schievan RR. A standard protocol for the quality control of
microbiology media. Int J Food Microbiol 1992;17:183-98.
12. Zoutman D, Fleming C, Richardson H. Compliance with
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July 2005 Basu et al – Quality Control of Culture Media 163

NCCLS approved standard M22-A2 for the bacteriologic media
quality assurance: a survey of 124 Ontario microbiology
laboratories. Diagn Microbiol Infect Dis 2002;42:29-34.
13. Jones RN, Krisher K, Bird DS. Results of the survey of the
15. Krisher K. Survey results: the use of the NCCLS standard M22-
quality assurance for the commercially prepared microbiology
media. Arch Pathol Lab Med 2003;127:661-5.
14. MacLowry JD, Edson DC, Dreskin R. CAP Microbiology
Resource Committee survey of commercially prepared media.
In: The role of Clinical Microbiology in Cost Effective Health
Care. Skokie III. Smith JW. 1985. p. 555-9.
A2 and Quality assurance for commercially prepared
microbiologic culture media by clinical laboratories. Clin
Microbiol Newsletter 2001;23:59-63.

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