Various
microbiological tests performed in dairy industry can be broadly categorized in
to following groups:
- Direct enumeration of Total Bacterial Count e.g. Direct Microscopic Count Estimation of number of viable bacterial cells e.g. Standard Plate Count
- Assessing the microbial metabolic activities e.g. Dye Reduction Test
- Detection of specific Contaminants e.g. Coliforms, Pathogens
- Estimation of biochemical changes or metabolites formed in dairy products as a result of microbial growth e.g. acidity, gas production, toxin production etc.
i)
Direct Microscopic Count (DMC) Method
The DMC method
enables rapid enumeration of bacterial cells along with their study of
morphology of the total bacterial count in milk and cream with minimum equipment.
It consists of examination of stained films of a measured volume of milk or
milk product (0.01 ml) spread over 1 cm2 area
and dried on a glass slide under microscope. Somatic cells, shapes and
arrangement of bacterial cells present in films can be easily and rapidly
visualized and recorded. The microbial morphology and arrangement give the clue
to possible cause of high count while high somatic cell indicates udder
infection e.g. mastitis. For determination of average number of bacterial cells
or clumps of cells about 5 to 50 microscopic fields are scanned (fewer the
number of cells, more fields to be scanned). The diameter of a field is
measured with the help of a stage micrometer to calculate microscopic factor (MF).
The DMC/ml is then calculated as follows:
DMC/ml=N x MF
Where N= Average
number of cells per field
MF= Microscopic
Factor
MF= Area of
Smear/Area of Microscopic x 1/Volume of milk (0.01 ml)
=10,000/3.1416 x r2
This technique is
very useful for screening of milk supplies on the receiving platform of a dairy
plant as well as for grading of milk. However, the limitation of this method is
that both dead as well as viable cells are counted.
ii)
Standard Plate Count (SPC) Method
In this method a
known quantity of milk sample is diluted to known degree and equal portions of
each dilution is poured in to a petriplate followed by addition of nutrient
agar medium, a technique known as pour plate method. The medium is allowed to
solidify after mixing the contents by gentle rotation of the plate. The organisms
present in the sample are expected to be trapped in the agar gel. The plates
are subsequently incubated at 37 C for 48 to 72 hours. In principle each organism
is expected to take up a separate position in the medium and grow in to a mass of cells
of a size sufficient enough to be counted by naked eyes,recognized as a colony
forming unit (cfu). Hence, a colony count performed at this stage represents
number of viable bacteria present in the given volume of milk sample. The major
limitations of this method is that it is time consuming and only those bacteria
which are capable of growing under given set of growth conditions (medium,
incubation temperature and period) and forming colonies can be counted. Determination
of microbiological quality of milk and milk products invariably involves
performing different plate counts. These include SPC, the Coliform count,and
the yeast and mold count. Techniques employed for plating are identical for these
tests though method of sampling and media may vary.
iii)
Dye Reduction Test
There are certain
dyes, which act as oxidation-reduction indicator. Bacteria consume dissolved
oxygen during their growth in milk and consequently reduce the OH to a level at
which these dyes are reduced and get decolorized. Such dyes can be employed to
assess the biochemical activity of bacteria and thus estimate number of
bacteria indirectly.
i)
Methylene Blue Reduction (MBR) Test
Methylene blue is a
dye, which remains blue in its oxidized state and turns colorless on its
reduction. This characteristic is put to use for estimation of bacterial load
of milk and milk products. When bacteria grow in milk they release hydrogen during
respiration, which is simultaneously accepted by methylene blue. As a result,
it is reduced to colorless or leuco compound.
The majority of
bacteria, both aerobic and facultative present in milk indulge in lowering of
oxidation-reduction potential of milk to such an extent that dye gets decolorized.
Hence greater the number of viable cells, shorter is the time taken to reduce
the dye. The result of this test is expressed in terms of time required for the
color of methylene blue to disappear at incubation temperature of 37o C.
This test renders
very useful information on general bacteriological quality of milk in a short
period and requires fewer apparatus. Limitations of this technique include
suitability only for unheated milk, no indication of type of organisms and incubation
temperature favorable only for mesophilic bacteria.
ii)
Resazurin Reduction (RR) Test
Resazurin is also
an OH indicator and hence is liable to be reduced by bacteria. Reduction of
blue dye takes place in two stages. First, the dye is irreversibly reduced to
resorufin undergoing through a series of colors ranging from blue to lilac,
mauve, purple and pink. During second stage, resorufin is reversibly reduced to
a colorless compound, dihydroresorufin.Various colors developed sequencely
during reduction of dye can be well compared with a standard resazurin disc
with the help of a small apparatus known as resazurin comparator. Results are
expressed in terms of standard resazurin disc number ranging from 6 to 0.The
time taken for the reduction of dye to a specific stage (disc number) or the
color change recorded on completion of incubation after a certain
period can be used as a scale for measurement of bacterial activity.The test is
carried out at incubation temperature of 370 C
for 10 minutes, I hour or till complete reduction.This test finds its
application in quick grading of milk (even faster than MBR Test).However,
reduction of dye is susceptible to light and confusion may arise in interpretation
of results due to the fact that besides bacteria, this dye is liable to be
reduced by leucocytes.
iv.
Coliform Test
The Coliform group
of bacteria (Escherichia, Enterobacter, Klebsella) includes gram-negative,
non-spore forming, aerobic and facultative rods capable of fermenting lactose
in to lactic acid and gas.As per American Public Health Association (APHA)
method, Coliforms in milk are detected by following scheme:
Presumptive
Coliform Test: One ml of milk sample or decimal dilution is poured to sterile
plates followed by addition of 10-15 ml of Violet Red Bile Agar (VRBA). The
content of plates is by gently rotating and tilting each dish and finally agar
is allowed to solidify and incubated at 320 C
for 24 h. Appearance of typically red colonies measuring about 0.5 mm is taken
as positive test.Alternatively fermentation tubes of 2% brilliant green lactose
bile (BGLB) broth are inoculated with sample (1.0 ml) and production of gas
after incubation of 48 h at 320 C is considered as
positive indication for presence of Coliforms in milk.
Confirmed
Test: A confirmed test of doubtful colonies form VRBA is carried out
by transferring each of five colonies to tubes of 2 % BGLB broth and observing
for gas production.
Completed
Test: Finally, material from typical colonies on solid media or from BGLB
broth tubes showing gas production is streaked on Eosine Methylene Blue Agar.
Coliforms form dark colonies or dark centered colonies with colorless peripheries
and red colonies on VRBA. Pure cultures so isolated should be able to produce
gas in fermentation tubes of lactose broth at 320C
within 48 h and microscopic examination should reveal only gram-negative,
non-spore forming
rods.
v.
Detection of Pathogens
Milk is a
favourable niche for pathogens and various pathogens found in milk have been
discussed in detail in unit 2. In this section, detection of two frequently encountered
pathogens viz. Salmonellae and Staphylococci have been discussed. Both of these
organisms are enterotoxigenic (produce enterotoxin). Being heat labile, though
these organism may be destroyed during heat treatment, yet their toxins survive
Majority of heart treatment. National and international microbiological standards
have specified limits for them in various dairy products.Staphylococci are gram
positive, catalase positive, coagulase positive (coagulate blood plasma) cocci
occurring singly or in clusters. The undiluted sample or its decimal dilution
of milk/milk product is analyzed by plate count method using selective media
(Trypticase soy broth, Baird Parker Agar, Vogel and Johnson agar, Staphylococci
medium No. 110). Appearance of typical black colonies on agar surface after
incubation for 48 h at 370 C is taken as a positive test.
Subsequently,
Coagulase test is performed to confirm their presence. Colonies are picked from
the agar plates, inoculated into brain heart infusion broth tube and 0.5 ml of
coagulase plasma is added before incubation at 370 C
for 6 h. The tubes are periodically observed for clot formation as a positive
reaction. Doubtful colonies may be further subjected to additional tests such
as catalase reaction, anaerobic utilization of glucose and mannitol,
susceptibility to lyostaphin etc.The detection
scheme for Salmonellae is elaborate. This organism is gram negative, may or may
not produce H2S. Presence of other related organisms e.g. Escherichia, Enterobacter,
Shigella and Proteus might prove interfering in interpretation of results.
These organisms are differentiated on the basis of reaction they exhibit on slants
of Triple Sugar Iron agar and appearance of characteristic colonies on the surface
of differential agar media as shown in below Tables
 |
| Appearance of typical colonies of salmonella on agar |
 |
| sugar iron or lysin iron agar |
vi. Yeast and Mould Count
Potato dextrose
agar (PDA) is the medium of choice for yeast and mould count as potato extract
promotes the growth of these organisms. The suppression of bacterial growth
which may otherwise interfere, is ensured by adjusting the pH of medium by
adding 10% tartaric acid. The plate count technique is used and plates are
incubated at 210 C for 2 to 5 days.
