CHAPTER 2:

MICROSCOPIC EXAMINATION OF
MICROORGANISM
CONTENTS

1. Microbial Analysis of food

 Food sampling
 Homogenization
 Serial Dilution
 Identification of Microorganisms

2. Microscopic examination of microorganism

MICROBIAL ANALYSIS OF FOOD
The quality of food:
By determining type and number of
microorganisms in food and by comparing this
standard value, we can evaluate quality of food

The storage condition: The sanitary condition of food:

By microbial analysis we can detect Microbial analysis helps to predict
whether food is stored in proper possible contamination of food.
condition or not.

The types of microorganisms in food: Evaluation of effectiveness:

By microbial analysis, we can detect Microbial analysis also evaluates the
pathogen or their toxic product if present in effectiveness of preservative methods
food. employed.

The quality of raw material:

It gives quality of raw materials used in food.
 STEPS IN MICROBIAL ANALYSIS OF FOOD

2. Techniques
3. Preparation 4. Transportation 5. Sampling 6. Sampling
1. Sampling for sample
of sample of sample report plan
collection
 1. FOOD SAMPLING

 Objective: The adequate condition of the sample/specimen received for examination are
important
 established sampling procedures must be applied uniformly.
 essential when pathogens or toxins are sparsely distributed within the food
 Samples should:
 be representative of whole lot of food - interpretations about a large consignment of food are
based on a relatively small sample of the lot
 be collected randomly from the lot.
 The principle is to avoid bias and to draw sufficient number of sample units
 Number of samples:
 If food is examined for Salmonella, usually co-samples are collected. 5 samples are needed for other
organisms.
 obtain at least 100 g for each sample unit
 original storage conditions maintained as nearly as possible
 Collect frozen samples in pre-chilled containers.
 Make a record for all samples of the times and dates of collection and of arrival at the laboratory
 2. TECHNIQUES FOR SAMPLE
COLLECTION:
 The proper sampling procedure; whether the food is solid, semisolid, viscous, or liquid
 submit samples to the laboratory in the original unopened containers
 containers too large - transfer representative portions to sterile containers under aseptic
conditions
 containers that are clean, dry, leak-proof, wide-mouthed, sterile, and of a size suitable

 For dry materials, use sterile metal boxes, cans, bags, or packets with suitable closures
 not to overfill bags or permit puncture by wire closure
 Identify each sample unit with a properly marked strip of masking tape
 Transport frozen or refrigerated products in approved insulated containers of rigid construction
 When the product is in big container: Steps in sample collection are:
 Outer surface of container is cleaned by washing or by 70% ethanol.
 Container is opened with the help of sterile cutting instrument.
 If the food is in bulk, the sample are collected from various places within container.
 In case of liquid sample, it is first agitated to form homogenous mixture and sample is taken.
 When the food is in small container:
 When the sample is in small container, they should be taken directly to the lab for analysis
 3. PREPARATION OF SAMPLE:

 For solid food sample: Solid food is mixed with sterile diluent in mechanical blender to
obtain homogenous suspension. For example, when 10gm food is added to 100ml diluent,
10-1 dilution is obtained.
 For liquid food sample: Liquid food is serially diluted to obtain 10-1, 10-2, 10-3 dilution.
HOMOGENIZATION
 Function- to make sure the microbe’s population is uniformly distributed.
 liquid form –
shake it by using your hand or with the help of vortex mixture.
 Other than liquid samples –
grinding or crunching in the mixing unit
 MIXING EQUIPMENT CRITERIA

 It’s able to homogenate the sample without increasing the sample temperature
 It’s able to separate and break the food sample without damaging the microorganisms cells
 It’s place no harm to the person using it
 Easy to wash, dry, use and sterilize
 Eg. stomacher
 homogenization will be done;

E.g : 50 grams of food sample will be

homogenate with 450 ml diluent

 should take place in the sterilized mixing unit for 2 minutes at 15000 up to 20000 rpm
 temperature of the sample should be maintained at low as room temperature along the
process
 THE DILUENTS

 the type of liquid used to dilute the food sample and turn the sample into
colloidal form
 must suitable with the type of food sample
 able to support the growth of the microbe cells in it
 should be no reaction occurs between the diluent and the components inside the
food sample
TYPE OF DILUENTS

 0.1 M Phosphate Buffer solution,

 0.1% bacteriology peptone solution.
 sterilized distilled water
 sterilized deionized water
 SERIAL DILUTION

 determine the number of cells in a bacterial culture

 cell numbers are very high in original sample
 samples must be diluted considerably to obtain reasonable counts
 done by repeatedly dilution
 A small amount of each of the diluted bacteria samples is then spread onto an agar plate
 The numbers of bacteria colonies that grow on each plate are counted
 "dilution factor"
 eg : 1 ml added to 9 ml gives a 10-fold dilution;
 Eg :1 ml added to 99ml gives a 100-fold dilution.
 the new sterilized pipette should be used

 When fixed amounts of this dilution series are mixed with an appropriate agar and incubated,
then different numbers of colonies will be obtained
 Aseptic techniques must be used!
EXAMPLE PROCEDURES OF SERIAL DILUTION IN
PLATING DILUTION

1
 Flame and loosen the lids of tubes 0
and 1.
 Transfer 1 ml of liquid from tube 0 to 2
plate -0, and also into tube 1.
 DISCARD THIS PIPETTE. Flame the
edge of tube 1.
 Seal and mix the contents gently.
 REPEAT THE SAME STEPS, 5 OR 6 TIMES,
MOVING ALONG THE CHAIN AS SHOWN IN THE
3 FLOW CHART BELOW.
PLATE COUNT DILUTION
PROCEDURE
 4. TRANSPORTATION OF SAMPLE:

 Multiplication and death of sample (microorganisms) should be prevented during transport

of sample.
 The sample should be delivered to lab immediately.
 Perishable and unfrozen sample should be cooled to 0-5oC and transported in insulated
container.
 Frozen product must be maintained in frozen condition until analysis.
 Sampling reports should be prepared and sent with sample.
 5. SAMPLING REPORT:

 The report should provide the following information:

 Name and address of person collecting sample.
 Date, time, place and purpose of sample collection.
 Nature of food
 Name of manufacturer, importer, seller, batch number, manufacture data, expiry
date etc.
 Method of sampling, size and no. of samples, temperature of product at the time
of sampling.
 Suggested test can also be given.
 6. SAMPLING PLAN:

 A sampling plan is a systematic way to assess the microbiological quality of foods.

 A sampling plan includes both the sampling procedure and the decision criteria.
 A quantity (“lot”) of product is produced, handled, and stored within a limited
time period under uniform conditions.
 It is impractical to examine all products. Instead of this, the number and size of
sample units from the lot are used for the analysis.
 The samples should be taken from the lot independently and randomly.
 In developing a sampling plan, a number of factors should be considered:
properties of food, production processes, storage conditions, associated risks,
targeted consumers, and limitations.
 Each food product should be considered individually.
 SAMPLING PLAN

• n is the number of sample units (packages, beef patties,

Microorganism and so forth) from a lot.
or group of • c is the maximum acceptable number or maximum
microorganism
s of concern. allowable number of sample units. When it exceeds the
microbiological criterion, the lot is rejected.
• m is the maximum number of microorganisms (criteria)
per gram; values above this level are either marginally
Sampling
Number of acceptable or unacceptable.
procedure sampling samples to be
• It is used to separate acceptable from
tested (n).
plan unacceptable foods in a two-class sampling plan or
elements • separate good quality foods from marginally
acceptable quality foods in a three-class sampling
plan.
• M is the quantity that is used to separate marginally
• acceptable (<m);
acceptable quality food from unacceptable food. It is
• marginally Microbiological used only in three-class sampling plans. Values above
acceptable Testing
(>m and <M);
limit(s), m and
method(s). M for any sample are unacceptable and indicate health
M:
• unacceptable (>M). hazard, sanitary indicators, and spoilage potential.
 SAMPLING PLAN ARE OF TWO TYPES:
Two class plan
 This plan is simplest and is used to ‘accept’ or ‘reject’
food product.
 A sampling plan for Salmonella is n=5, c=0,
 where n=5 means that five individual samples of the lot are
examined microbiologically for the presence of Salmonella
and c =0 means that all five units must be free of the
bacteria for the lot to be acceptable. If any unit is positive
for Salmonella, the entire lot is rejected
Samples may contain coliforms with a sampling plan n=5,
c=2. According to this plan, if three or more of the five unit
samples contain coliforms, the entire lot would be rejected. If
up to 100 coliforms per gram are allowed in two of the five
units, the sampling plan would be n=5, c=2, m=102. After the
five units have been examined for coliforms, the lot is
acceptable if no more than two of the five units contain as
many as 102 coliforms per gram, but is rejected if three or
more of the five units contain 102 coliforms per gram.
Three class plan
 A three-class sampling plan is used to indicate
acceptable/marginally acceptable/unacceptable foods.
 Assume that for a given food product, the standard
plate count shall not exceed 106 colony forming unit
(cfu) g-1 (M) or be higher than 105 cfu g-1 from three or
more of the five units examined.
 The specifications become n=5, c =2, m=105, M=106. If
any of the five units exceeds 106 cfu g-1, the lot is
rejected (unacceptable).
 If not more than c sample units give results above m,
the lot is acceptable.
 If two of the five units exceed m and do not exceed M,
the lot is marginally accepted.
 EXAMPLE: MICROBIAL ANALYSIS OF MILK
 In general market milk is sampled in the container in which
it is sold.
 Sterile plunger is used for sampling and 10ml sample is
taken. The collecting sample is put in sterile screw capped
tube and immediately send to lab.
 Before performing test, the sample is mixed by inverting up
and down for more than 10 times.
 For milk bottle, the top is flamed, cap removed with flame
forceps.
 For plastic packets, the top and corner of packet is swabbed
with spirit.
 After allowing to air dry, the swabbed area is cut with
sterile scissors.
 The collected milk is tested by methylene blue test
resazurin test, MPN test.
 Three routine tests prescribed for milk are:
 Plate count at 32oC: serial dilution is made and pour
plating is done from tubes of 10-2– 10-8 dilution.
 For raw milk, DMC is usually done.
 Coliform test: It is done by MPN
 Mesophilic aerobic bacteria in Milk:
 Raw milk should not exceed total plate count = 2x 105/ml
 Pasteurized milk should not exceed total plate count = 2x
104/ml
 At 37oC, coliform = 0 (best)
 At 42oC, fecal coliform = 0 (best)
 For dried milk,
 Mesophilic aerophilic bacteria = n=5, c=2, m= 5X 104, M =
5×105
 Coliform= n=5, c=2, m=2, M=102
 Staphylococcus aureus = n=5, c=1, m=10, M=102
 Salmonella = n=10, c=0, m=0
ADVANCED TECHNIQUES IN FOOD
MICROBIOLOGY
 Several physical, chemical, and immunological rapid (advanced) methods are
available to overcome disadvantages of conventional methods.
 Rapid microbiological methods can be grouped into three classes:
 (i) labor-saving methods in which results are obtained in the traditional time
frames (such as laser counting of agar plates);
 (ii) methods in which results are obtained between 6 and 24 h (such as radiometry,
impedance method, and microcalorimetry); and
 (iii) very rapid methods in which results are obtained in less than an hour (such as
direct epifluorescence, bioluminescence, and Limulus amoebocyte lysate assay).
RAPID TEST

 Advances in biotechnology have introduced new techniques for food testing that are
faster and more sensitive than traditional methods.
 These techniques are collectively known as rapid methods, including miniaturized
biochemical tests, specialized media and substrates, and DNA- and antibody-based
detection assays.
 Rapid methods have advantages that results are obtained in minutes or hours, and the
methods are typically less labor intensive and may provide increased sensitivity and
specificity. Rapid methods are often more sensitive than conventional methods.
 Disadvantages with respect to cost (often expensive) and need for technician proficiency.
 Table 1. Examples of the application of nucleic acid-based methods for the detection
of various foodborne pathogens present in food samples.
https://www.frontiersin.org/files/Articles/122692/fmicb-05-00770-r4/image_m/fmicb-05-
00770-t001.jpg
MICROSCOPIC EXAMINATION OF
MICROORGANISM
 MICROSCOPY

 The science of investigating small objects using such an instrument (microscope) – microscopy

 The microscope is at tool that allows us to see the structures and tissues that comprise organisms
in very fine detail.

 Ability to observe structures too small for the unaided eye (ex: cells and previously unknown
creatures).

 Magnification: produce image larger than actual structure

 Resolution: ability of the microscope to show two close objects separately

 MICROSCOPY
 Possible to understand function of a structure on the basis of its
microscopic morphology.
 Many theories basic to biologic sciences were developed after invention of
the microscope.
 The basic microscope system is a light microscope or a bright field
microscope which allows visible light passes directly through its lenses
until it reaches the eye
 the compound microscope - a two lens system, with the objective lens
nearer the object and the ocular lens nearer the eye
• three objective lenses namely the low-
power, high power and oil immersion
lenses
• magnify an object about 10, 40 and 100
times (magnification symbol by X)
GRAM STAINING

 as an aid in the identification and classification of isolates

 various staining methods for bacteria, by far the most important is the Gram
stain.
 Bacteria are grouped on the basis of their Gram reaction either positive or
negative
 GRAM STAINING SOLUTIONS
1. Crystal violet
2. Iodine
3. Alcohol
4. Safranin
 Gram-positive bacteria retain the primary dye,
crystal violet (CV), following the application of the
mordant, iodine (I). Mordant is a substance that
increases the cells’ affinity for a stain.

 The iodine and crystal violet form a complex (CV-I)

within the peptidoglycan. When a decolorizer is
applied to the cells, the CV-I complex remains within
the cell, making it appear dark purple to blue.

 In Gram-negative cells, following the application of

the crystal violet and iodine, the CV-I complexes are
not trapped within the peptidoglycan.

 Application of the acid-alcohol decolorizer

dehydrates the outer cellular membrane, and also
dissolves the lipids leaving holes in the membrane
and effectively washing or removing the CV-I
complex from the cells.

 The cells appear colorless. Therefore, a counter

stain, safranin, is applied, to make the cells
distinctly visible (either red or pink).

 Gram-positive organisms that have lost cell wall

integrity because of anti-biotic treatment, old age,
or action of autolytic enzymes may allow the crystal
violet to wash out with the decolorizer and appear
Gram-variable, with some cells staining pink
and others staining purple.
ELECTRON MICROSCOPE

 Different types of electron microscope;

1. Transmission electron microscope (TEM)
An electron beam passes through a very thin section of material
Electron pass through some part and creates image

2. Scanning Electron Microscope (SEM)

A narrow beam of electron is scanned in series of lines across the surface of
specimen
The reflected/emitted electrons from the surface are collected by a detector and
converted into electrical signal – used for 3D image build up
SEM
TRANSMISSION ELECTRON MICROSCOPE (TEM)

TEM
 LIGHT VS ELECTRON MICROSCOPE
Light microscope Electron microscope

Material can be prepared easily Prep. of material takes longer times

Living material can be examined Living material cannot survive in the vacuum inside
electron microscope

Movement can be observed No movement can be observed

Colours can be seen (both natural or artificial) Only monochrome images are produced

Large field of view (2 mm) Only small field of view (about 100 um)

Poor resolution (about 0.25 um) Very good resolution (about 0.25 nm)

Up to 600x magnification Up to 500 000x magnification

 Scanning Electron Transmission Electron Microscopes (TEM)
Microscopes (SEM)

Electron stream Fine, focused beam Broad beam

Image taken Topographical/surface Internal structure
Resolution Lower resolution Higher resolution
Magnification Up to 2,000,000 times Up to 50,000,000 times
Image dimension 3-D 2-D
Thin and thick samples
Sample thickness Ultrathin samples only
okay
Penetrates sample No Yes
Sample restriction Less restrictive More restrictive
Less preparation
Sample preparation More preparation required
required
Cost Less expensive More expensive
Speed Faster Slower
Operation Easy to use More complicated; requires training