10. MICROBIOLOGICAL ANALYSIS
10.1 Characteristics of indicator organisms
To assure a safe water supply, it is critical to monitor for the presence of possibile
pathogens. However, it would be expensive and time consuming to check the water supply
for all of them, instead, an indicator organism is used to assay for fecal contamination. The
detection of indicator bacteria is preferred over direct pathogen detection because the
former are considered to be normal, non-pathogenic intestinal inhabitants that are present
in feces and wastewater in much higher numbers than pathogenic microorganisms and
because they are technically easier to detect and quantitate than pathogens. Present
standards for the sanitary quality of water, foods and other materials, with respect to fecal
contamination, are expressed in terms of concentrations of indicator bacteria.
Indicator organisms must have four properties to be useful for water analysis:
- the only natural environment of the microbe should be in association with
feces and it should always be present.
- it should not grow outside of its natural environment.
- the bacterium should survive longer than the most viable pathogen, but not
so long so that historical events are detected.
- it should be easy to detect.
Coliforms come closest to fulfilling all these criteria and are the standard indicator
organisms used to test for the biological pollution of water. The word "coliform" has been
used to describe various genera of the family Enterobacteriaceae that ferment lactose.
Total coliforms are operationally defined as "all facultative anaerobic, gram-negative, nonspore-forming, rod-shaped bacteria that ferment lactose with gas formation within 48 hr. at
35oC." The fecal coliform group are considered to be a more specific indicator of fecal
contamination because the total coliform group probably includes more bacteria of nonfecal origin. The basis for this separation is a higher incubation temperature of 44.5OC, at
which presumably coliforms of only fecal origin will grow. Coliforms from non-fecal,
environmental sources are generally incapable of growing at this elevated temperature.
Thus, fecal coliforms can be defined as gram-negative, non-sporeforming, rod-shaped
bacteria which ferment lactose with the production of gas at 44.5oC within 24 hours. Or,
using the newer chromogenic substrate media, the fecal coliforms would hydrolyze ONPG
(o-nitrophenyl-beta-D-galactoside) at 44.5oC within a specified time (24-28 hours). The
fecal coliform test is applicable to investigations of surface and ground water pollution,
sewage treatment systems and general monitoring of natural waters for sanitary quality,
including recreational and shellfish waters. It is also used in addition to the coliform test in
the examination of potable waters.
In recent years a new approach to detecting total and fecal coliform bacteria has
become widely used that is not based directly on lactose fermentation but instead is based
on detecting the expression of beta-galactosidase activity. The expression of this
enzymatic activity is detected by the ability of the bacteria to hydrolyze and utilize a
chromogenic substrate rather than lactose. This substrate is o-nitrophenyl-beta-Dgalactoside (ONPG). When the coliform bacteria hydrolyze ONPG, o-nitrophenyl is
released, which imparts a yellow color to the originally colorless medium, thereby
demonstrating the presence of coliform bacteria. So, the definition of coliforms is now
broadened to include any bacterium that hydrolyzes ONPG in a selective culture medium
under defined growth conditions of incubation temperature (35 oC) and time (24-28 hours).
Most recently, there is increased interest in detecting E. coli exclusively as the lactosefermenting "coliform" that invariably indicates fecal contamination. An early approach to
this effort was the use of four biochemical tests (called the IMViC tests) to separate E. coli
from other lactose-fermenting Enterobacteriaceae:. It was later shown that this approach is
not very reliable, and alternative methods were developed:
- Indole test - detects indole production from tryptophane. E. coli is positive (+);
many other coliforms are negative.
- Methyl Red test - detects acid production in the medium; intended to distinguish
between type of fermentation (mixed acid versus butylene glycol). E. coli is (+)
and some other coliforms are (-).
- Voges-Proskauer test - detects acetoin, an intermediate in the butylene glycol
pathway. Acetoin is oxidized to diacetyl under alkaline conditions in the presence
of air, and when reacted with creatine, it forms a pink color. E. coli is (-) and
some other coliforms are (+).
- Citrate utilization as sole carbon source. E. coli is (-) and many other coliforms
are (+).
Standards for clean, safe water vary, depending upon the waters intended use.
Drinking water and the water in swimming pools must be of the highest purity. There can
be no more than one positive sample (>1 coliform/100 ml) in 40 samples tested in a month
and the concentration of fecal coliforms must be zero.
Another usefull measure is heterotrophic plate count. It includes all of the microorganisms that are capable of growing in or on a nutrient-rich solid agar medium. Two
incubation temperatures and times are used: 37 °C for 24 hours to encourage the growth
of bacteria of mammalian origin, and 22 °C for 72 hours to enumerate bacteria. The main
purpose of colony counts is in comparing the results of repeated samples from the same
source. If number of colonies increase substantially for the short time, there may be cause
for concern.
10.2 Selecting a bacteriological analytical technique
Two specific methods was developed and standardized for microbiological assesment
of water. The first is Multiple Fermentation Tube Technique (also known as Most Probable
Number Technique). In this method measured portions of a water sample are placed in
test-tubes containing a culture medium, and then are incubated for a standard time at a
standard temperature. The second technique is Membrane Filter Technique. A measured
volume of sample is passed through a fine filter that retains bacteria. The filter is then
placed on culture medium and incubated.
10.3 Multiple fermentation tube technique
The multiple tube coliform test has been a standard method for determining coliform
group bacteria since 1936. It involves adding the water sample to a set of tubes, each of
which contains either lactose broth or lauryl tryptose broth and an inverted tube. The
tubes are then incubated at 35 ± 0.5º C for 24 to 48 hours (fecal coliforms are incubated in
a water bath at 44.5 ± 0.2º C). If gas production is observed in an inverted tube after
incubation, the sample contains total coliforms.
The multiple-tube fermentation technique is applicable to the analysis of fresh, salt, or
brackish waters as well as muds, sediments, and sludges. When multiple tubes are used
in the fermentation technique, results of the examination of replicate tubes and dilutions
are reported in terms of the Most Probable Number (MPN) of organisms present. This
number, based on certain probability formulas, is an estimate of the mean density of of
coliforms in the sample. Coliform density provides the best assessment of water treatment
effectiveness and the sanitary quality of untreated water.
The precision of each test depends on the number of tubes used. The most satisfactory
information will be obtained when the largest sample inoculum shows no gas in all or a
majority of the tubes. Bacterial density can be estimated by the formula given or from the
table using the number of positive tubes in multiple dilutions. The number of sample
portions selected will be governed by the desired precision of the result. MPN tables are
based on the assumption of a Poisson distribution (random dispersion). However, if the
sample is not adequately shaken before the portions are removed or if clumping of
bacterial cells occurs, the MPN value will be an underestimate of the actual bacterial
density.
Presumptive Phase:
Use lauryl tryptose broth in the presumptive portion of the multiple-tube test.
a. Reagents and culture medium: Add dehydrated products to water, mix thoroughly,
and heat to dissolve. pH should be 6.8 +/- 0.2 after sterilization. Before sterilization,
dispense sufficient medium, in fermentation tubes with an inverted vial, to cover inverted
vial at least one-half to two-thirds after sterilization. Alternatively, omit inverted vial and add
0.01 g/L bromcresol purple to presumptive medium to determine acid production, the
indicator of a positive result in this part of the coliform test. Close tubes with metal or heatresistant plastic caps.
b. Procedure:
1.Arrange the fermentation tubes in rows of five tubes each in a test tube rack. The
number of five-tube rows and the sample volumes selected depend upon the quality and
character of the water to be examined. For potable water use five 20-mL portions, ten 10mL portions, or a single bottle of 100 mL portion; for nonpotable water use five tubes per
dilution (of 10, 1, 0.1 mL, etc.). Shake sample and dilutions vigorously about 25 times.
Inoculate each tube in a set of five with replicate sample volumes (in increasing decimal
dilutions, if decimal quantities of the sample are used). Mix test portions in the medium by
gentle agitation.
2. Incubate inoculated tubes or bottles at 35 +/- 0.5oC. After 24 +/- 2h swirl each tube or
bottle gently and examine it for heavy growth, gas, and acidic reaction (shades of yellow
color) and, if no gas or acidic growth has formed, reincubated and reexamine at the end of
48 +/- 3h. Record Presence or absence of heavy growth, gas, and acid production. If the
inner vial is ommited, growth with acidity signifies a positive presuptive reaction.
c. Interpretation: Production of gas or acidic growth in the tubes or bottles within 48
+/- 3h constitutes a positive presumptive reaction. Submit tubes with a positive
presumptive reaction to the confirmed phase. The absence of acidic growth or gas
formation at the end of 48 +/- 3h of incubation constitutes a negative test. An arbitrary 48-h
limit for observation doubtless excludes occasional members of the coliform group that
grow very slowly.
Confirmed Phase
a. Culture Medium: Use brilliant green lactose bile broth fermentation tubes for the
confirmed phase. Add dehydrated ingredients to water, mix thoroughly, and heat to
dissolve. pH should be 7.2 +/- 0.2 after sterilization. Before sterilization, dispense, in
fermentation tubes with an inverted vial, sufficient medium to cover inverted vial at least
one-half to two-thirds after sterilization. Close tubes with metal or heat-resistant plastic
caps.
b. Procedure: Submit all primary tubes or bottles showing heavy growth, any amount
of gas, or acidic growth within 24h of incubation to the confirmed phase. If active
fermentation or acidic growth appears in the primary tube earlier than 24h, transfer to the
confirmatory medium, preferably without waiting for the full 24h period to elapse. If
additional primary tubes or bottles show active fermentation or acidic growth at the end of
a 48h incubation period, submit these to the confirmed phase. Gently shake or rotate
primary tubes or bottles showing gas or acidic growth to resuspend the organisms. With a
sterile metal loop 3 mm in diameter, transfer one loopful of culture to a fermentation tube
containing brilliant green lactose bile broth. Remove and discard applicator. Repeat for all
other positive presumptive tubes. Incubate the inoculated brilliant green lactose bile broth
tube for 48 +/- 3 h at 35 +/- 0.5oC. Formation of gas in any amount in the inverted vial of
the brilliant green lactose bile broth fermentation tube at any time within 48 +/- 3 h
constitutes a positive confirmed phase. Calculate the MPN value from the number of
positive brilliant green lactose bile tubes.
c. Alternative procedure: Use this alternative only for polluted water or wastewater
known to produce results consistently. If all presumptive tubes are positive in two or more
consecutive dilutions within 24 h, submit to the confirmed phase only the tubes of the
highest dilution (smallest sample inoculum) in which all tubes are positive and any positive
tubes in still higher dilutions. Submit to the confirmed phase all tubes in which gas or acidic
growth is produced only after 48 h
Completed Phase
To establish the presence of coliform bacteria and to provide quality control data, use
the completed test on at least 10% of positive confirmed tubes. Double confirmation into
brilliant green lactose bile broth for total coliforms and EC broth for fecal coliforms may be
used. Consider positive EC broth elevated temperature (44.5oC) results as a positive
completed test response. Parallel positive brilliant green lactose bile broth culture with
negative EC broth cultures indicate the presence of nonfecal coliforms and must be
submitted to the completed test procedures to validate the presence of coliforms.
a. Culture media and reagents: Add ingredients to water, mix thoroughly, and heat to
dissolve. pH should be 6.8 +/- 0.2 after sterilization. Before sterilization, dispense in screwcapped tubes. After sterilization, immediately place tubes in an inclined position so that the
agar will solidify with a sloped surface. Tighten screw caps after cooling and store in a
protected, cool storage area.
b. Procedure:
1. Using aseptic technique, streak one LES Endo agar or MacConkey agar plate from
each tube of brilliant green lactose bile broth showing gas, as soon as possible after the
observation of gas. Streak plates in a manner to insure presence of some discrete
colonies separated by at least 0.5 cm. Observe the following precautions when streaking
plates to obtain a high proportion of successful isolations if coliform organisms are present:
(a) Use a sterile 3-mm-diam loop or an inoculating needle slightly curved at the tip; (b) tap
and incline the fermentation tube to avoid picking up any membrane or scum on the
needle; (c) insert end of loop or needle into the liquid in the tube to a depth of
approximately 0.5cm; and (d) streak plate for isolation with curved section of the needle in
contact with the agar to avoid a scratched or torn surface. Flame the loop between second
and third quadrants to improve colony isolation. Incubate plates (inverted) at 35 +/- 0.5oC
for 24 +/- 2h.
2. The colonies developing on LES Endo agar are defined as typical ( pink to dark red with
a green metallic surface sheen); atypical (pink, red, white, or colorless colonies without
sheen) after 24 h incubation; or negative (all others). Typical lactose-fermenting colonies
developing on MacConkey agar are red and may be surrounded by an opaque zone of
precipitated bile. From each plate pick one or more typical, well-isolated coliform colonies
or, if no typical colonies are present, pick two or more colonies considered most likely to
consist of organisms of the coliform group, and transfer growth from each isolate to a
single-strength lauryl tryptose broth fermentation tube and onto a nutrient agar slant. (The
latter is unnecessary for drinking water samples.)
Estimation of Bacterial Density
a. Precision of Fermentation Tube Test: Unless a large number of sample portions is
examined, the precision of the fermentation tube test is rather low. For example, even
when the sample contains 1 coliform organism/mL, about 37% of 1-mL tubes may be
expected to yield negative results because of random distribution of the bacteria in the
sample. When five tubes, each with 1 mL sample, are used under these conditions, a
completely negative result may be expected less than 1% of the time. Even when five
fermentation tubes are used, the precision of the results obtained is not of a high order.
Consequently, exercise great caution when interpreting the sanitary significance of
coliform results obtained from the use of a few tubes with each sample dilution, especially
when the number of samples from a given sampling point is limited.
b. Computing and Recording of MPN: To calculate coliform density, compute in
terms of the Most Probable Number. The MPN values are given in Table 1 (as an
example). Included in this table is the 95% confidence limits for each MPN value
determined. If the sample volumes used are those found in the tables, report the value
corresponding to the number of positive and negative results in the series as the MPN/100
mL or report as total or fecal coliform presence or absence. When the series of decimal
dilutions is different from that in the table, select the MPN value from the table for the
combination of positive tubes and calculate according to the following formula: MPN
value(from table) x (10/largest volume tested) = MPN/100mL
95%
Confidence
# of tubes giving positive reaction
MPN Index/100mL
Limits (approximate)
out of 10 of 10mL each
Lower Upper
0
less than 1.1
0
3.0
1
1.1
0.03
5.9
2
2.2
0.26
8.1
3
3.6
0.69
10.6
4
5.1
1.3
13.4
5
6.9
2.1
16.8
6
9.2
3.1
21.1
7
12.0
4.3
27.1
8
16.1
5.9
36.8
9
23.0
8.1
59.5
10
greater than 23.0
13.5
infinite
When more than three dilutions are used in a decimal series of dilutions, use the results
from only three of these in computing the MPN. To select the three dilutions to be used in
determining the MPN index, choose the highest dilution that gives positive results in all five
portions tested (no lower dilution giving any negative results) and the two next succeeding
higher dilutions. Use the results at these three volumes in computing the MPN index. In
the examples given below, the significant dilution results are shown in boldface. The
number in the numerator represents positive tubes; that in the denominator, the total tubes
planted; the combination of positives simply represents the total number of positive tubes
per dilution:
Example
1
mL
0.1
mL
0.01
mL
0.001
mL
Combination
positives
of MPN
mL
a
5/5
5/5
2/5
0/5
5-2-0
5000
b
5/5
4/5
2/5
0/5
5-4-2
2200
c
0/5
1/5
0/5
0/5
0-1-0
20
index/100
In c, select the first three dilutions so as to include the positive result in the middle dilution.
10.4. Membrane filter technique
The Membrane Filter (MF) Technique was introduced in the late 1950’s as an
alternative to the Most Probable Number (MPN) procedure for microbiological analysis of
water samples. The MF Technique offers the advantage of isolating discrete colonies of
bacteria, whereas the MPN procedure only indicates the presence or absence of an
approximate number or organisms (indicated by turbidity in test tubes).
The MF Technique is also used for microbial monitoring in the pharmaceutical,
cosmetics, electronics, and food and beverage industries. The MF Technique is used in
these industrial labs to monitor the presence of microorganisms in process waters and
final product.
In the Membrane Filter (MF) Technique, a vacuum pulls 100 mL of water sample
through a 47 mm membrane filter held in place by a filter-holding device. Total coliform
and other bacteria are retained on the filter. The filter is then placed on a special medium
which allows the growth of total coliform and incubated at 35 ± 0.5º C for 22 to 24 hours. If
a total coliform-like colony is observed on the membrane, the lab should make sure that it
is a total coliform by using another test.
The MF Technique provides presence or absence information within 24 hours.
Advantages of the MF Technique
•
•
•
•
•
•
•
Permits testing of large sample volumes.
Reduces preparation time as compared to many traditional methods.
Allows isolation and enumeration of discrete colonies of bacteria.
Provides presence or absence information within 24 hours.
Effective and acceptable technique. Used to monitor drinking water in government
laboratories.
Useful for bacterial monitoring in the pharmaceutical, cosmetics, electronics, and
food and beverage industries.
Allows for removal of bacteriostatic or cidal agents that would not be removed in
Pour Plate, Spread Plate, or MPN techniques.
The technique is unsuitable for natural waters containing very high levels of suspended
material, sludges and sediments, all of which could block the filter before an adequate
volume of water has passed through. When small quantities of sample (for example, of
sewage effluent or of grossly polluted surface water) are to be tested, it is necessary to
dilute a portion of the sample in sterile diluent to ensure that there is sufficient volume to
filter across the entire surface of the membrane.
Procedure
1. Add absorbent pads to sterile Petri dishes for the number of samples to be
processed. Sterile pads may be placed in the Petri dishes with sterile forceps or
with an automatic dispenser.
2. Soak the pads with nutrient medium. Nutrient medium may be dispensed with a
sterile pipette or by carefully pouring from an ampoule or bottle. In all cases, a slight
excess of medium should be added (e.g. about 2.5 ml). Immediately before
processing a sample, drain off most of the excess medium, but always ensure that
a slight excess remains to prevent the pad drying during incubation. Note:
Absorbent pads soaked in liquid medium may be replaced by medium solidified by
agar. In this case, Petri dishes should be prepared in advance and stored in a
refrigerator.
3. Sterilise the tips of the blunt-ended forceps in a flame and allow them to cool.
4. Carefully remove a sterile membrane filter from its package, holding it only by its
edge.
5. Place the membrane filter in the filter apparatus, and clamp it in place. If the
apparatus has been disinfected by boiling, ensure that it has cooled down before
inserting the membrane filter.
6. Mix the sample by inverting its container several times. Pour or pipette the desired
volume of sample into the filter funnel. This volume should normally be chosen in
the light of previous experience. If the volume to be filtered is less than 10 ml, it
should be made up to at least 10 ml with sterile diluent so that the sample will be
distributed evenly across the filter during filtration.
7. Apply a vacuum to the suction flask and draw the sample through the filter;
disconnect vacuum.
8. Dismantle the filtration apparatus and remove the membrane filter using the sterile
forceps, taking care to touch only the edge of the filter.
9. Remove the lid of a previously prepared Petri dish and place the membrane, grid
side uppermost, onto the pad (or agar). Lower the membrane, starting at one edge
in order to avoid trapping air bubbles.
10. Replace the lid of the Petri dish and mark it with the sample number or other
identification. The sample volume should also be recorded. Use a wax pencil or
waterproof pen when writing on Petri dishes.
11. If membranes are going to be incubated at 44 or 44.5 °C, the bacteria on them may
first require time to acclimatise to the nutrient medium. After processing samples
from areas of temperate climate, leave each Petri dish at environmental
temperature for 2 hours before placing it in the incubator. Samples from areas of
tropical climate may be incubated immediately.
12. Maintain the Petri dish in a humid atmosphere (e.g. in a plastic bag or in a small
container with a moist pad in the base) and incubate it either in an incubator or in a
weighed canister in a water bath. This ensures that the pad does not dry out during
the incubation period.
13. The incubation periods and temperatures required for each culture medium are
listed in Table 1. Characteristics of total coliform and thermotolerant coliform
colonies grown on the various culture media are described in Table 2.
14. After incubation, count the colonies. Express the results as number of colonies per
100 ml of sample. Where smaller volumes have been used, results are calculated
from the following formula: No. of colonies per 100 ml = [(No. of colonies)/(volume
filtered)] × 100. The colonies counted at this stage are presumed to be coliform
bacteria (presumptive results).
Table 1. Culture media for membrane filtration
Medium
Uses
Lactose
TTC
agar with
Tergitol 7
Total or
thermotolera
nt
coliforms
Lactose
agar
with
Tergitol 7
Total or
thermotolera
nt
coliforms
Membrane
enrichment
with
Teepol
broth
Total or
thermotolera
nt
coliforms
Membrane
lauryl
sulphate
broth
Total or
thermotolera
nt
coliforms
Endo
medium
Total
coliforms
only
LES Endo
medium
Total
coliforms
only
Incubation
temperature
18-24 hours at 35 ±
0.5°C or 37 ± 0.5°C for
total
coliforms
and
1824hours at 44 ± 0.25°C
or 44.5 ± 0.25°C for
thermotolerant
coliforms
Remarks
Adjust
pH
before
sterilisation. Filter TTC
supplement to sterilise.
Tergitol
supplement
sterilised by autoclaving.
Supplements of Tergitol
and TTC to be added
aseptically. Prepared plates
have max. shelf-life of 10
days. Store in dark.
18-24 hours at 35 ± Prepared plates have max.
0.5°C or 37 ± 0.5°C for shelf-life of 10 days. Store
prepared plates at 4°C.
total
coliforms
and
1824hours at 44 ± 0.25°C
or 44.5 ± 0.25°C for
thermotolerant
coliforms
pH
before
18-24 hours at 35 ± Check
0.5°C or 37 ± 0.5°C for sterilisation
total
coliforms
and
1824hours at 44 ± 0.25°C
or 44.5 ± 0.25°C for
thermotolerant
coliforms
pH
before
18-24 hours at 35 ± Check
0.5°C or 37 ± 0.5°C for sterilisation
total
coliforms
and
1824hours at 44 ± 0.25°C
or 44.5 ± 0.25°C for
thermotolerant
coliforms
35-37 °C
Basic fuchsin may be a
carcinogen. Also requires
ethanol. Do not autoclave.
Prepared medium has a
shelf-life of 4 days. Store
prepared medium at 4 °C in
the dark.
35-37 °C
Basic fuchsin may be a
carcinogen. Also requires
ethanol. Do not autoclave.
Prepared medium has a
shelf-life of 2 days. Store
prepared
medium at 4 °C in the dark.
MFC
Thermotoler
ant
coliforms
44 °C
Do not autoclave. Discard
unused medium after 96
hours. Rosalic acid stock
solution has a maximum
shelflife of 2 weeks. Check
pH before sterilisation.
Store prepared medium at
2-10 °C.
Table 2. Colony characteristics following analysis by the membrane filtration method
Medium
Lactose TTC agar
with Tergitol 7
Lactose agar with
Tergitol 7
Membrane
enriched
Teepol broth
Membrane lauryl
sulphate broth
Endo agar or broth
LES Endo agar
MFC medium
Total coliforms at 35 or 37 °C
Thermotolerant
coliforms
at 44 or 44.5 °C
Yellow, orange or brick red Same as total coliforms
coloration with yellow central at
halo in the medium under the 35 or 37 °C
membrane
Yellow central halo in the Same as total coliforms
medium under the Membrane
at
35 or 37 °C
Yellow colour extending on to Same as total coliforms
the membrane
at
35 or 37 °C
Yellow colour extending on to Same as total coliforms
the membrane
at
35 or 37 °C
Dark red colour with golden- (not applicable)
green metallic sheen
Dark red colour with golden- (not applicable)
green metallic sheen
(not applicable)
Blue colonies
Confirmatory tests
For the examination of raw or partly treated waters, presumptive results may be
adequate but, in certain other circumstances, it is important to carry out confirmatory tests
on pure subcultures.
To confirm the membrane results for total coliforms, each colony (or a representative
number of colonies) is subcultured to tubes of lactose peptone water and incubated at 35
or 37 °C for 48 hours. Gas production within this period confirms the presence of total
coliforms.
To confirm thermotolerant coliforms and E. coli on membranes, whether incubated at
35, 37 or 44 °C, each colony (or a representative number of colonies) is subcultured to a
tube of lactose peptone water and a tube of tryptone water. Tubes are incubated at 44 °C
for 24 hours. Growth with the production of gas in the lactose peptone water confirms the
presence of thermotolerant coliforms. Confirmation of E. coli requires the addition of 0.20.3 ml of Kovac’s reagent to each tryptone water culture. Production of a red colour
indicates the synthesis of indole from tryptophan and confirms the presence of E. coli.
10.5 Quality assurance
Quality assurance encompass a number of principles and cautions which ensure
continuous quality and reproducibility of results. Analytical quality control that apply to
microbiological laboratories comprises the preparation and control of laboratory
consumables (media and dilution solutions, membrane filters and pads, plasticand
glassware), and also a proper laboratory techniques and manuveurs. Special concern is
given to monitoring of laboratory equipment.
Incubators, refrigerators and freezers should be cleaned at least once a month. The
manufacturers’ instructions should include advice on cleaning and may recommend
suitable detergents and disinfectants. Water-baths may need more frequent cleaning to
control bacterial growth.
One should allways keep in his mind that some detergents and other agents used for
the cleaning of laboratory glassware may influenced the growth of bacteria and interfere
final results of analyses. Samples of glassware should be microbiologicaly examined
regularly, for example once a month, if washing procedures and products are always the
same. If procedures change, however, or new products are introduced, additional checks
should be made. Laboratory plastics may also contain inhibitory residues and each new
batch of plasticware should be checked.