Chapter Eight Labratory Analysis of Food

Chapter eight
Laboratory analysis of foods
1 Mastewal E. (BSc, MPH) Monday, January 2, 2023

Learning objectives
At the end of this chapter students will be able to
 Discuss about food sampling technique
 Demonstrate Physical tests
 Demonstrate Microbiological tests
 Identify the different types of media
 Discuss proximate analysis

Activities in analysis of food
• Basic activities involved in analysis of food
products:
Sample preparation and collection of representative
sample
Analysis using appropriate methods and instruments and
interpretation of results

First step of food sample analysis
[Sampling and sample preparation]

Food Sampling and Preparation of Sample
Definition of sampling:
Obtaining a portion, or sample, that is representative of the whole is

referred to as sampling.
 The total quantity from which a sample is obtained is called the

population.
Adequate sampling technique helps to ensure that sample quality

measurements are an accurate and precise estimate of the quantity of
the population.
5 Monday, January 2, 2023
 By sampling only a fraction of the population, a quality estimate
 can be obtained more quickly and with less expense , personnel and time
than if the total population were measured.
 The sample is only an estimate of the value of the population, but with
proper sampling technique, it can be a very accurate estimate.

During food sampling assure the following
Quality control
Representative sampling [Uniformity and Homogeneity]
Sampling quality
 To increase the quality of the sample
What is aseptic techniques?

• For dry materials, use sterile metal boxes, cans, bags, or
packets with suitable closures.
• Take care not to overfill bags or permit puncture by wire

closure.
• Do not use a felt pen on plastic because the ink might penetrate
the container.
• Deliver samples to the laboratory promptly with the original

storage conditions.
• When collecting liquid samples, take an additional sample as a

temperature control.
• Check the temperature of the control sample at the time of
collection and on receipt at the laboratory.
• Make a record for all samples of the times and dates of collection
and of arrival at the laboratory.
• Dry or canned foods that are not perishable and are collected at
ambient temperatures need not be refrigerated.
• Transport frozen or refrigerated products in approved insulated

containers of rigid construction so that they will arrive at the
laboratory unchanged.
• Collect frozen samples in pre chilled containers.

• sterile sampling equipment ( stainless steel spoons, forceps,
spatulas, and scissors in an autoclave or dry-heat oven).
• Use of a propane torch or dipping the instrument in alcohol to

disinfect
• Use containers that are clean, dry, leak proof, wide mouthed,
and of a size suitable for samples .
• Containers such as plastic jars or metal cans that are leak-proof

may be hermetically sealed.
• Whenever possible, avoid glass containers, which may break

and contaminate the food product.
• Place containers in a freezer long enough to chill them
thoroughly.
• Cool refrigerated samples, except shellfish and shell stock, in ice

at 0-4°C and transport them with suitable refrigerant capable
of maintaining the sample at 0-4°C until arrival at the
laboratory.
• Do not freeze refrigerated products.
• Unless otherwise specified, refrigerated samples should not be

analyzed more than 36 h after collection.
Representative sample collection
 Representative sampling should be taken from the source.
 How we collect representative sampling?
 In small group, discus how we can collect representative
sampling?
Sample must be taken from a number of locations within the
population to ensure it is representative of the whole
population.
• For liquids in small containers, this can be done by shaking
prior to sampling.
• Liquids may be sampled when sampling from a large volume
of liquid, aeration ensures a homogeneous unit.
• by pipetting, pumping, or dipping.
• However, when mixing is impossible and samples are obtained
by probing from several points at random within the
population
NB:
1. A sample, consisting of a specified number of sample units
(usually five) drawn at random from each lot, shall be taken.
2. Each sample unit shall consist of at least 100 ml or g or cm2

unless stipulated in the method.
3. Collect original unopened container wherever possible.

4. If the product is in bulk, several sample units can be
collected from one container, while ensuring that the

total number of sample units are not collected from one
container.
• More than one sample unit may also be collected from

large institutional or bulk containers.
• A sample unit will consist of more than one container

when the lot consists of containers smaller than 100 ml or
eg. four 25 ml or g containers in each sample unit.
Minimum Number of Primary Samples to be taken
Products packaged in bulk, 1
which can be assumed to be (A lot may be mixed by
well mixed or homogeneous grading or
manufacturing
process)
Products packaged Weight of lot, kg
in bulk, which may
not be well mixed <50 3
or homogeneous. 50-500 5
>500 10
Number of cans, cartons, containers in the
lot
1-25 1
26-100 5
> 100 10
Lot:
• A batch or production unit which may be identified by the
same code. When there is no code identification, a lot may be
considered as
(a) that quantity of product produced under essentially the same

conditions, at the same establishment and representing no
more than one day's production; or,
(b) the quantity of the same kind of product from one and the
same manufacturer available for sampling at a fixed location.
Sampled Food items
 Prepared foods
 Grain
 Flour
 Liquid
 Semi liquid foods
 Beverage
 Vomits or faeces from victims
 Food utensils
 Food handlers’ hand, finger nail,cloths

Types of tests
1. Physical or organo-loptic test:
 Using sense organ
 Tests like color, flavour, odour, texture, infestation by insects, etc
2. Chemical and toxicological test:

 adulterants, excessive cleaning agents, or even indications of processing
abuse in the form of chemical by-products
 Poisons, and microbial toxins
3.Microbiological test:
 Microorganisms caused spoilage, food borne out break
 Norovirus, Escherichia coli (causes traveler's diarrhea), Listeria, Salmonella
and Staphylococcus aureus
How to analyze
i) Quantitative analysis
• Serial decimal dilution
• Aerobic plate count
• Pour plate count
• Total viable count
• Most Probable Number (MPN) method
• Yeast and Molds count

ii) Qualitative analysis
Presence or absence of a specified microorganism

e.g.
• Salmonella sp.
• E. coli
• V. cholerae O1
Scope of physical test
 Color
 Texture
 Water activity (Aw
 Moisture
 Appearance, tightness
 Count and size
 Granulation/particle size
 Temperature
 Viscosity, Weight Monday, January 2, 2023

23
Water activity(Aw)
 Water activity (Aw), which determines how much water is available
for microbial growth, can affect food odor, color, flavor, texture, and
shelf-life.
 The chance for microbial spoilage is reduced with lower Aw.
 The Aw can be measured using a water activity meter
 Methods for moisture measurement, such as the Karl Fischer
method, distillation method, dielectric method, hydrometry method,
refractometry method, oven drying methods

Mastewal E. (BSc, MPH)
Structure
 The food structure can influence food texture.
 Scanning electron microscopy, X-ray micro-CT, light and confocal
laser scanning microscopy, etc.) to qualitatively and quantitatively
determine the structure of food.
Texture
 Food textural attributes, such as hardness, fracturability,
compressibility, shear force, and springiness, are important attributes
25 forMastewal
food sensory.
E. (BSc, MPH)
Monday, January 2, 2023
Particle size
 Particle size determined by machine sieve screening, laser diffraction, and
static light scattering.
Color
 The color of foods is an important criterion for food quality evaluation.
 Color measurement is used to predict visual and chemical changes in
foods.
 The most widely used method for color measurement is the Hunter “L a b”
system, where “L” represents lightness (100)/darkness (0); “a” is positive

with redness and negative for greenness; “b” measures yellowness when
"positive" and blueness when "negative". Monday, January 2, 2023
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Viscosity and consistency
 Viscosity measures the flow resistance in soups, beverages, shake mixes,
and syrup solutions.

 Consistency measures the distance that a sample flows in a predefined
time interval.
 Viscosity and consistency can be measured via the electromagnetic
measurement approach, where viscosity (in centipoise) and

temperature (in °C) are measured simultaneously.
 Viscosity or flow properties can be measured using rheological testing
equipment.

PH
 The pH is an indicator of the amount of acid or base present in a food.
 The pH level can not only affect the growth of microorganisms, but
also affect the flavor, color, and texture.

 The pH of food products can be measured using a pH meter.

Microbiological testing
 Microbiological testing of food is the examination of the microscopic
organisms in food.
 These organisms could be single cell, multiple cell or without cell.
 Microbiology includes various sub-disciplines like Virology,
Mycology, Parasitology and Bacteriology.

Why microbial food analysis is important?
Reasons for microbial food analysis.
• To meet certain set standards
• To estimate the shelf-life of the product
• To determine quality of the food
• For public health purposes

What organisms to look for ?
i) Indicator organism(s)
Definition:
an indicator organism or group of organisms is one whose

numbers in a product reflect the success or failure of "good
manufacturing practices".
Coliform group of microorganisms and Escherichia coli are

31 commonly used as indicator organisms. Monday, January 2, 2023
ii) Index organism
Definition:
an index organism is one whose presence implies the possible

occurrence of a similar but pathogenic organism. e.g. E. coli ፣
Salmonella sp.
iii) Food poisoning organisms
iv) Infectious microorganisms
v) Spoilage organisms
Common microbiological Test Methods
1. Culture Media
2. Immunoassay
3. Polymerase chain reaction

1.Culture media
 A special medium that is use to detect different types of microorganisms by
culturing or growing.
 Media are solidified most frequently by agar, or gelifying agent.
 Agar media when molten are used for the preparation of pour plates,
whereas gel state used for streaking or spreading plate.

Types of culture media
1. BASAL MEDIA. It used for growth (culture) of bacteria that do not
need enrichment of the media.
 They usually contain a source of carbon, energy (often glucose), salts,
amino acids, and vitamins

 Examples: Nutrient broth, nutrient agar and peptone water.
 It used to enumerate and isolate certain types of microorganisms.
2. ENRICHED MEDIA.
 The media are enriched usually by adding blood, serum or egg.
 Examples: Enriched media are blood agar and Lowenstein-Jensen

media. Streptococci grow in blood agar media.
35 Mastewal E. (BSc, MPH)
3. SELECTIVE MEDIA
 These media favour the growth of a particular bacterium by inhibiting the
growth of undesired bacteria.
 Examples: MacConkey agar, Lowenstein-Jensen media, tellurite media
4. INDICATOR (DIFFERENTIAL) MEDIA.
An indicator is included in the medium. A particular organism causes
change in the indicator. E.g blood, neutral red, tellurite. Examples: Blood
agar and MacConkey agar are indicator media. Monday, January 2, 2023
36
6. TRANSPORT MEDIA
 These media are used when specie-men cannot be cultured soon after
collection.
Examples: Cary-Blair medium, Amies medium, Stuart medium.
7. STORAGE MEDIA
 Media used for storing the bacteria for a long period of time.
 Examples: Egg saline medium, chalk cooked meat broth

Common media in routine use
 Nutrient Broth. - 500 g meat
 Blood Agar - 5- 10% sheep or horse blood is added to melted agar at 45-
50°C
 MacConkey Agar - It contains agar, peptone, sodium chloride, bile salt,
lactose and neutral red.

Key Features of culture media
 Both liquid and solid culture media are employed.
 Microscopes are usually used to detect microbes in cultures
 Biochemical and serological techniques are used to differentiate
various organisms.
 Both qualitative and quantitative results of microorganisms can be
obtained using cultural methods.

 Quantitative analysis is only possible using solid culture media
 Time to attain results can range from twelve hours to more than a
week.

Culture media

2. Immunoassay
 It is used to measure the concentration of a macromolecule in a solution
via using an antibody or immunoglobulin.
 The detected macromolecule from this method is protein
 Urine or serum -- are analytes measured following immunoassay test
methods

2. Key features of Immunoassay
• Antibodies are used to detect and identify specific proteins, which are
predicted to be unique to the target microorganism.
 Both qualitative and quantitative results can be possible, but methods are
usually detected/not-detected (qualitative) type tests
 Time to obtain results can range from 24 hours to 48 hours.

3. Polymerase chain reaction (PCR)
 PCR is a very recent and revolutionary method developed
 Today, it is used in medical and biological research labs
 A PCR test can recognize pieces of DNA or RNA, which are expected to
be unique to the target microorganism.
 PCR is based on using the ability of DNA polymerase and can generate
billions of copies of a specific DNA sequence.

Key features of Polymerase chain reaction (PCR)
 Selected sections of DNA or RNA can be reproduced using the PCR
technique.
 Both qualitative and quantitative results can be possible, but PCR
methods are usually detected/not-detected (qualitative) type test
 Test methods can be sensitive and rapid, predominantly when paired with
a cultural pre-enrichment.
 Test results can be obtained within 24-48 hours

Detection of microorganisms in food
The four basic methods employed for “total” numbers are:
1. Standard plate count for viable cells
2. Most probable number as statistical determination of viable cells
3. Dye reduction technique to estimate number of viable cells that
possess reducing capabilities
4. Direct microscopic count for viable and non viable cells.

1. Conventional Standard Plate Count
• portions of food samples are blended or homogenized
• Serially diluted in an appropriate diluent, plated in or onto a suitable agar

medium
• incubated at an appropriate temperature for a given time (48 hrs.)
• all visible colonies are counted by use of a Quebec or electronic counter.
• determine the numbers of viable cells or colony-forming units (cfu) in a

food product.
Serial dilution method
Types of Standard plate count (SPC) method
1. Pour plate technique
• In this method, food is firstly serially diluted in appropriate diluent.
• Then, measured volume of sample from diluted tube is placed in

petriplate.
• Melted agar at 44-45oC is mixed with it.
• After homogenous mixing of sample with melted agar, it is kept for

solidification.
• Then the petri plates are incubated at appropriate time and temperature.
• Plate containing colonies between 30-300 is selected and number of

colonies are counted.
2. Spread plate technique
• In this method, appropriately diluted sample is placed on the surface of solidified agar.
• Then the drop of sample is spread over agar surface using bent glass rod.
• Plate is incubated for sufficient time and temperature, then number of colonies are
counted.
3. Streak plate technique

• In this technique, a transfer loop is used to spread the specific volume of specimen over
a surface of solidified agar.
• The transfer is done by calibrated loop of specific volume.
• Sometimes, selective and differential media can be used to select growth of specific
organism.
1. Pour plate technique
• Number of organisms in original food sample is calculated
by the following formula:
• Colony forming units (CFU/ml) = (number of colonies
/volume of sample ) x dilution factor

1.1. Membrane Filters
• retain bacteria (generally a pore size 0.45 μm) but allow water or
diluent to pass are used.
• Following the collection of bacteria upon filtering a given

volume, the membrane is placed on an agar plate or an absorbent
pad saturated with the culture medium of choice and incubated
appropriately.
• Following growth, the colonies are enumerated.

• Alternatively, a direct microscope count can be made.
• organisms collected on the membrane are viewed and counted

microscopically following appropriate staining, washing, and
treatment of the membrane to render it transparent.
• This method is especially suited for samples that contain low

numbers of bacteria.
1.2. Microscope Colony Counts
• Involve the counting of micro colonies that develop in agar layered

over microscope slides.
• 2 ml of melted agar are mixed with 2 ml of warmed homogenate (eg.

milk) and, after mixing, 0.1 ml of the inoculated agar is spread over
a 4 cm2 area.
• Following incubation, drying, and staining, micro colonies are

counted with the aid of a microscope.
1.3. Agar Droplets
 This method is reliable to enumerate microorganisms present in meats and
vegetables
the food homogenate is diluted in tubes of melted agar (at 45◦C).
For each food sample, three tubes of agar are used, the first tube
being inoculated with 1 ml of food homogenate.
After mixing, a sterile capillary pipette (ideally delivering 0.033

ml/drop) is used to transfer three drops (0.1 ml) to the bottom of an
empty Petri dish.
With the same capillary pipette, three drops (0.1 ml) from the first
tube are transferred to the second tube,
and after mixing, another 0.1-ml droplets is placed next to the first.
 This step is repeated for the third tube of agar.
 Petri plates containing the agar droplets are incubated for 24 hours,
and colonies are enumerated.
This method is about three times faster, and 24-hour incubations

gave counts equal to those obtained after 48 hours by the
conventional plate count.
Dilution blanks are not required, and only one Petri dish per sample
is needed.
1.4. Aerobic colony count
• Enumeration of viable aerobic bacteria (psychrophilic, mesophilic

and/or thermophilic bacteria) in foods.
Principle
• Estimates the number of viable aerobic bacteria per g or
mL of product.
• A portion of the product is mixed with a specified agar
medium and incubated under specific conditions of time
and temperature.
• count the visible colonies.
2. Most Probable Numbers
It is used to detect coliform bacteria which are used as indicator of
faecal contamination.
Dilutions of food samples are prepared as for the SPC.
Three serial aliquots or dilutions are then planted into 9 or 15 tubes of

appropriate medium for the three- or five-tube method, respectively.
Numbers of organisms in the original sample are determined by use of

standard MPN tables.
The method is statistical in nature, and MPN results are generally

higher than SPC results.
It is not a precise method of analysis; the 95% confidence intervals
for a three-tube test range from 21 to 395.
When the three-tube test is used, 20 of the 62 possible test

combinations account for 99% of all results,
whereas with the five-tube test, 49 of the possible 214 combinations

account for 99% of all results.
 advantages
I. It is relatively simple.
II. Results from one laboratory are more likely than SPC
results to agree with those from another laboratory.
III. Specific groups of organisms can be determined by use

of appropriate selective and differential media.
IV. It is the method of choice for determining fecal

coliform densities.
Drawbacks
Needs large volume of glassware especially for the five-tube method,
lack of opportunity to observe the colonial morphology of the organisms,

and
its lack of precision.

3. DYE REDUCTION
Two dyes (methylene blue and resazurin) are commonly

employed to estimate the number of viable organisms.
To conduct a dye-reduction test,

 properly prepared supernatants of foods are added to standard
solutions of either dye for reduction
• from blue to white for methylene blue; and
• from blue to pink for resazurin.
The time for dye reduction to occur is inversely proportional to the

number of organisms in the sample.
The suggested classification of the result
• Class 1. Excellent, not decolorized in 8 hours.
• Class 2. Good, decolorized in less than 8 hours
but not less than 6 hours. Methylene Blue
• Class 3. Fair, decolorized in less than 6 hours Reduction Test
but not less than 2 hours.
• Class 4. Poor, decolorized in less than 2 hours.
Color of Sample: Quality of Milk
• Blue (no color change): Excellent
• Blue to deep mauve: Good
The Resazurin Test
• Deep mauve to deep pink: Fair
• Deep pink to whitish pink: Poor
• White: Bad
Dye-reduction tests have a long history of use in the dairy
industry for assessing the overall microbial quality of raw milk.
advantages
 simple, rapid, and inexpensive;
 only viable cells actively reduce the dyes.
Disadvantages
not all organisms reduce the dyes equally,
 not applicable to food specimens that contain reductive

enzymes.
4. Direct Microscopic Count (Dmc)
 A rapid method for the initial morphological identification and count
of bacteria and molds.
Making smears of food specimens or cultures onto a microscope slide
Staining with an appropriate dye, and viewing and counting cells with
the aid of a microscope.
DMCs are most widely used in the dairy industry for assessing the
microbial quality of raw milk and other dairy products.
Used also to the rapid microbiological examination of other food

products, such as dried and frozen foods.
Method
Add 0.01 ml of a sample to a 1-cm2 area on a microscope
slide
fixing, defatting of sample, and staining,
Count / enumerate micro colonies.
Advantages
it is faster than most other methods because incubation
period is not required
cell morphology can be assessed,
Disadvantages
• Both viable and nonviable cells are enumerated,
• food particles are not always distinguishable from

microorganisms,
• microbial cells are not uniformly distributed relative to single
cells and clumps,
• some cells do not take the stain well and may not be counted,
and
• DMC counts are invariably higher than counts by SPC.
5.Microbiological examination of surfaces
1. Swab / swab – rinse methods
2. Contact plate
3. Agar syringe / agar sausage methods
4. Direct surface method
5. Sticky film
1. Swab / swab – rinse methods
• Used for the microbiological examination of surfaces in the food

and dairy industries, hospitals and restaurants.
• Either cotton or calcium alginate swabs are used
• The exposed area (1in2,1 cm2 or 3 cm2) is rubbed or swabbed

thoroughly with a moistened swab.
• The exposed swab is returned to its holder (test tube) containing

a suitable diluent and stored at refrigerator temperatures until
plated.
• The diluent should contain a neutralizer.
• If necessary. When cotton swabs are used, the organisms must be

dislodged from the fibers.
• When calcium alginate swabs are used, the organisms are released into
the diluent upon dissolution of the alginate by sodium
hexametaphosphate.
• The organisms in the diluent are enumerated by a suitable method such

as SPC.
• Any of the culture media may be used to test specifically for given
groups of organisms.
• Other method of swab-rinse
• 1.5 ml of fluid is added to a flat surface,
• swabbed for 15 seconds over a 3-cm2 area,
• collected in microliter pipettes.
• The fluid may be surface or pour plated using plate count agar or
selective media.
• Drawbacks
• uneven, and heavily contaminated surfaces.
• The ease of removal of organisms depends on the texture of the

surface and the nature and types of biota.
2. Contact Plate
• The replicate organism direct agar contact (RODAC)

method employs special Petri plates, which are poured with
15.5–16.5 ml of an appropriate plating medium.
• When the plate is inverted, the hardened agar makes direct

contact with the surface.
• Once exposed, plates are covered and incubated, and the

colonies enumerated.
3. Agar Syringe/“Agar Sausage” Methods
• A 100-ml syringe or a hollow cylinder will be filled with agar.
• A layer of agar is pushed beyond the end of the barrel by

means of the plunger
• And pressed against the surface to be examined.
• The exposed layer is cut off and placed in a Petri dish,
• followed by incubation and colony enumeration.

4. Direct Surface agar plating
• A melted agar is poured onto the surface or utensil to be

assessed.
• Upon hardening, the agar mold is placed in a Petri dish and

incubated.
• Enumeration of CFUs.
5. Sticky Film
• Pressing sticky film or tape against the surface to be examined
• pressing the exposed side on an agar plate.
• incubation and enumeration of CFUs.
• It was shown to be less effective than swabs in recovering

bacteria from wooden surfaces.
• An adhesive tape method has been employed successfully to

assess microorganisms on meat surfaces
Bioassay and related methods for the detection of biologically
active organisms or their toxins
1. Mouse lethality
2. Suckling (infant) mouse
3. Rabbit and mouse diarrhhea
4. Monkey feeding
5. Kitten (cat) test
6. Rabbit and guinea pig skin test

1. Mouse Lethality
To test for botulinal toxins (Clostridium botulinum, Clostridium

perfringens and others) in foods
Appropriate extracts are made and portions are treated with

trypsin (for toxins of non proteolytic Clostridium botulinum
strains).
Pairs of mice are injected intraperitoneally (IP) with 0.5 ml of

trypsin-treated and untreated preparations.
Untreated preparations that have been heated for 10 minutes at
100oC are injected into a pair of mice.
All injected mice are observed for 72 hours for symptoms of

botulism or death.
Mice injected with the heat preparations should not die because
the botulinal toxins are heat labile.
2. Suckling (Infant) Mouse
 For Escherichia coli enterotoxins and for some other food borne pathogens.
Typically, mice are separated from their mothers and given oral doses of the
test material consisting of 0.05–0.1 ml with the aid of hypodermic needle.
The animals are usually held at 25oC for 2 hours and then killed.
The entire small intestine is removed, and the relative activity of
test material is determined by the ratio of gut weight to body
weight (GW/BW).
The test material may also be injected percutaneously directly in

the stomach.
The intestines may be examined visually for dilation and fluid

accumulation.
Giannella found the following GW/BW ratios for E.coli
enterotoxins:<0.074=negative test; 0.075–0.082=intermediate
(should be retested); and >0.083 = positive test.
The investigator found the day-to-day variability among various E.

coli strains to range from 10.5% to 15.7% and about 9% for replicate
tests with the same strain.
A GW/BW of 0.060 was considered negative for E. coli by Mullan.
 In studies with E. coli , Wood et al. treated as positive GW/BW

ratios that were >0.087.
3. Rabbit and Mouse Diarrhea
Rabbits and mice have been employed to test for diarrhea

genic activity of some food borne pathogens with the
required dose of pathogen to cause infection.
After some days, feces of mice were examined for signs of

diarrhea.
The quantity of enterotoxin is achieved by ascertaining

the ratio of intestinal weight to total body weight.
Young pigs have been used in a similar way.

4. Monkey Feeding
The use of rhesus monkeys (Macaca mulatta) to assay

staphylococcal enterotoxins
This monkey is the most sensitive animal to staphylococcal

enterotoxins next to human.
When enterotoxins are to be assayed by this method, young

rhesus monkeys weighing 2–3 kg are selected.
The food homogenate, usually in solution in 50-ml quantities, is

administered via stomach tube.
The animals are then observed continuously for 5 hours.
Vomiting in at least two of six animals denotes a positive response.
Rhesus monkeys have been shown to respond to levels of

enterotoxins A and B as low as approximately 5µg per 2–3 kg of
body weight.
5. Kitten (Cat) Test
As an assay for staphylococcal enterotoxins.
The original test employed the injection of filtrates into the

abdominal cavity of very young kittens (250 – 500 g).
The most commonly used method consists of administering

the filtrates IV and observing the animals continuously for
symptoms.
When cats weighing 2–4 kg are used, positive responses occur

in 2–6 hours.
Emesis has been reported to occur with 0.1 and 0.5µg of staphylococcal
enterotoxin A (SEA) and SEB per kilogram of body weight.
The test tends to lack the specificity of the monkey-feeding test.
 because staphylococcal culture filtrates containing other byproducts may

also induce symptoms.
6. Rabbit and Guinea Pig Skin Tests
The skin of these two animals is used to assay toxins for at least
two properties.
The vascular permeability test is generally done by use of albino

rabbits weighing 1.5–2.0 kg.
Typically, 0.05–0.1 ml of culture filtrate is inoculated

intradermally (ID) in a shaved area of the rabbit’s back and sides.
From 2 to 18 hours later, a solution of Evans blue dye is
administered and 1–2 hours are allowed for permeation by the
dye.
The diameters of two blue zones are measured and the area
approximated by squaring the average of the two values.
 Areas of 25 cm are considered positive.

2
Proximate analysis
 Proximate analysis refers to the determination of the major constituents of
food
 it is used to assess if a food is within its normal compositional parameters
or somehow been adulterated
 This method partitioned nutrients in food into total protein, crude fat,
carbohydrate, ash, and moisture and expressed as the percentage
content in the food, respectively

Proximate analysis

Determination of Moisture Content by Hot Air Oven Method
Methods
 Homogenize the sample thoroughly in a domestic mixer.
 Weigh about 5g in a clean dried petri-dish pre-dried at 98°C for 60
minutes.
 Dry the sample over a period ranging from 2 to 3 hours in a hot air oven
at 100±1°C.
 Cool in a dessicator and weigh until it reaches a constant value.
 The percent moisture content can be calculated from the difference
between the initial sample weight (WI) and the final sample weight after
drying. (WD).
Determination of Moisture Content by Hot Air Oven Method

Determination of Crude Ash Content Using Muffle Furnace
 Ash is the residue obtained after incineration of the dry material at high
temperatures and appears as greyish white coloured powder.
 Heat a platinum crucible to 600°C in a muffle furnace for 1 hour, cool in a
desiccator and weigh (W1).
 Weigh accurately 5g of the dried sample (W2) in to a crucible and heat at
low flame by keeping on a clay triangle to char the organic matter.
 Keep the charred material inside the previously set muffle furnace and
heat for 6 to 8 hours to greyish white ash at 600°C.

Mastewal E. (BSc, MPH)
Determination of Crude Ash Content Using Muffle Furnace
 Cool the crucible in a desiccator and weigh (W3).
 Cool and weigh after heating the crucible again for further 30 minutes
to confirm completion of ashing

Determination of Total Protein by Micro Kjeldahl Method
 Weigh 2 g of homogenized wet sample in to a Micro Kjeldahl flask
of 100 ml capacity.
 Add a few glass beads and a pinch of digestion mixture (a mixture
of Copper sulphate and Potassium sulphate in the ratio of (1:8) and

10 ml of concentrated Sulphuric acid.
 Digest over a burner till solution turns colourless.
 To the digested solution in digestion flask add distilled water in
small quantities with shaking and cooling till the addition of water
does not generate heat.
95
Determination of Total Protein by Lowry’s Method
 Transfer quantitatively in to a 100 ml standard flask and make up the
volume and pipette 5 ml of the made up solution and transfer to the
reaction chamber of the Micro Kjeldahl distillation apparatus. Rinse
down with distilled water.
 Add 2 drops of Phenolphthalein indicator and 40% Sodium hydroxide till
the indicator changes to pink. Distill for 4 minutes and absorb the
ammonia liberated in 2% Boric acid containing a drop of Tashiro’s
indicator and determine the amount of ammonia by treating with N/50

9
Sulphuric acid
Determination of Total Protein by Lowry’s Method
Optical Density (O.D)

Determination of Crude Fat by Soxhlet Method
 Weigh 10 g of dry sample into a thimble and keep a cotton plug on top it.
 Place the thimble in a Soxhlet apparatus and add ½ volumes of Ether into a
pre-weighed flat-bottom flask (W2) and distilled for 16 hours (Cool the
apparatus and filter the solvent into a pre-weighed conical flask (W2).
 Rinse the flask of the apparatus with small quantities of ether.
 Remove Ether by evaporation and dry the flask with fat at 80-100°C, cool
in a desiccator and weigh (W3)

98
Determination of Crude Fat by Soxhlet Method

Determination of Total carbohydrate by Furfural Colorimetric Method
 Weigh about 30 mg of sample into a 20 ml centrifuge tube.
 Heat in a boiling water bath for 30 minutes with 4 ml 10% Tri Chloro
acetic acid (TCA), and 30 mg of Silver sulphate.
 After centrifuging, transfer the clear supernatant and the subsequent
washings of the residue with the TCA solution to a 25 ml graduated flask
and make up to the volume.

Determination of Total carbohydrate by Furfural Colorimetric Method
 Take 2 ml aliquots in duplicates and carefully layer over a 6 ml of
concentrated Sulphuric acid in a boiling tube.
 Quickly agitate to mix the contents thoroughly and heat for 6.5 minutes in
a vigorously boiling water bath.
 Cool rapidly to room temperature and measure the optical density at 520
nm. Run blanks with each batch of analysis and use glucose to observe the
standard curve

Thank you!

Chapter Eight Labratory Analysis of Food

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Chapter eight

Laboratory analysis of foods

1 Mastewal E. (BSc, MPH) Monday, January 2, 2023

At the end of this chapter students will be able to

 Discuss about food sampling technique

 Demonstrate Physical tests

 Demonstrate Microbiological tests

 Identify the different types of media

 Discuss proximate analysis

2 Mastewal E. (BSc, MPH) Monday, January 2, 2023

3 Mastewal E. (BSc, MPH) Monday, January 2, 2023

[Sampling and sample preparation]

4 Mastewal E. (BSc, MPH) Monday, January 2, 2023

Obtaining a portion, or sample, that is representative of the whole is

 The total quantity from which a sample is obtained is called the

Adequate sampling technique helps to ensure that sample quality

than if the total population were measured.

proper sampling technique, it can be a very accurate estimate.

6 Mastewal E. (BSc, MPH) Monday, January 2, 2023

What is aseptic techniques?

• Take care not to overfill bags or permit puncture by wire

• Deliver samples to the laboratory promptly with the original

• When collecting liquid samples, take an additional sample as a

• Transport frozen or refrigerated products in approved insulated

• Collect frozen samples in pre chilled containers.

• Use of a propane torch or dipping the instrument in alcohol to

• Containers such as plastic jars or metal cans that are leak-proof

• Whenever possible, avoid glass containers, which may break

• Cool refrigerated samples, except shellfish and shell stock, in ice

• Do not freeze refrigerated products.

• Unless otherwise specified, refrigerated samples should not be

 Representative sampling should be taken from the source.

 How we collect representative sampling?

 In small group, discus how we can collect representative

2. Each sample unit shall consist of at least 100 ml or g or cm2

3. Collect original unopened container wherever possible.

collected from one container, while ensuring that the

• More than one sample unit may also be collected from

• A sample unit will consist of more than one container

(a) that quantity of product produced under essentially the same

 Semi liquid foods

 Vomits or faeces from victims

 Food handlers’ hand, finger nail,cloths

2. Chemical and toxicological test:

• Aerobic plate count

• Pour plate count

• Total viable count

• Most Probable Number (MPN) method

• Yeast and Molds count

Presence or absence of a specified microorganism

 Water activity (Aw

 Count and size

 Viscosity, Weight Monday, January 2, 2023

 The chance for microbial spoilage is reduced with lower Aw.

 The Aw can be measured using a water activity meter

 Methods for moisture measurement, such as the Karl Fischer

method, distillation method, dielectric method, hydrometry method,

refractometry method, oven drying methods

 The food structure can influence food texture.

 Scanning electron microscopy, X-ray micro-CT, light and confocal

laser scanning microscopy, etc.) to qualitatively and quantitatively

determine the structure of food.