FOOD MICROBIOLOGY LABORATORY
Stage The platform on which
REVIEWER
PART 1: MICROSCOPE
PART OF FUNCTIONS
MICROSCOPE
1. Body Tube Where the eyepieces are
dropped in. Also, it
connects the eyepieces to
the objective lenses.
2.Nosepiece his circular structure is
where the different
objective lenses are
screwed in. To change the
magnification power, simply
rotate the turret
3. Objective The lens closest to the
Lenses specimen that first receives
the rays from the specimen
(the object) and forms the
image in the focal plane of
the eyepiece
4.Stage Clips Clips that are attached to
the stage and retain the
slide.
5.
slides and specimens are
placed for viewing.
6.Diaphragm The diaphragm or iris is
located under the stage and
is an apparatus that can be
adjusted to vary the
intensity, and size, of the
cone of light that is
projected through the slide
7. Illuminator A steady light source (110
volts in the US) that shines
up through the slide.
Mirrors are sometimes used
in lieu of a built-in light. If
your microscope has a
mirror, it is used to reflect
light from an external light
source up through the
bottom of the stage
8.Eye Piece/ Otherwise referred to as an
Ocular Lense ocular, the eyepiece is the
lens nearest to your eye.
Total magnification of a
microscope is determined
by the sum of the eyepiece
magnification multiplied by
that of the objective lens.
9. Arm The part of the microscope
that connects the eyepiece
tube to the base. 
10.Coarse This is the knob on the side
Focus of the microscope that
moves the objective lens up
and down. It is used in
conjunction with the fine
focus.
11. Fine Focus A knob used to fine-tune
the focus of a specimen in
conjunction with the coarse
focus.
12. Base A microscope is typically
composed of a head or
body and a base. The base is
the support mechanism.
PART 2: MATERIALS USED IN microbiological cultures can be used in
MICROBIOLOGY determination, egg incubation, heated storage,
food and beverage testing, and pharmaceutical
Apparatus and Equipment Commonly Used
testing
in the Microbiological Laboratory
Laboratory Drying Oven- evaporation,
Test tubes- Rimless tubes 160 to 180 mm in
sterilization, temperature testing, and for
length and 16 to 18 mm in diameter are
incubating temperature sensitive experiments.
preferred.
Triple beam balance - is generally used to weigh
Erlenmeyer flasks- The culture medium
media, for amounts up to 15 grams, glazed
incubated in an Erlenmeyer flask is in contact
weighing paper can be used to hold the powder
with the air over a large surface area. This
medium.  For bigger amounts, a small beaker
favors growth in aerobic cultivation.
may be used. 
Roux bottles- quadrangular or rounded in
Equipment of the Food Microbiology
profile, suitable for incubation in a horizontal
Research Laboratory
position.
Bioscreen-C Automated Growth Curve Analysis
Petri dishes- each 1 cm in height forms a
System- for measurement of growth of
culture vessel. The medium is poured into the
bacteria, molds, yeasts, or spores (it can create
smaller dish, which is about 9 cm in diameter,
up to 200 growth curves from 200 samples at
the larger one being used as a loosely-fitting lid.
one time)
Beakers- used for mixing, stirring and heating
miniVIDAS® instrument (multiparametric
liquids.
immunoassay system-Biomerieux) for food
Durham tubes- small glass tubes sealed at one pathogen testing and measurement of
end and open at the other. To indicate gas Staphylococcal enterotoxin
formation in a culture, it is placed with the open
A Thermo Scientific Forma 902 -86°C upright
end facing downwards in a test tube containing
Freezer for long-term storage of bacteria and
liquid medium.
samples
Pipettes- commonly used pipettes are
Microscopes: Zeiss Axion Star (with camera)
graduated ones, 1 mL full scale.
and Axion Star Plus.
Glass rods- used for spreading suspensions of
Microbiological safety cabinet Top Safe class II,
microorganisms on solid media.
type1.2, BIOAIR
Straight wires (needles) and wire loops for
Cooling incubators from 0°C up to 99.9 °C with
inoculation- made of platinum or chromium
12 different programs, Friocell 111, MMM
nickel and are fixed in a holder. Depending on
group and Incubators from 5°C above ambient
the use of the loop, its diameter may vary
temperature up to 99.9°C, Incucell 111, MMM
between 2 mm and 8 mm.
group
Autoclave- to sterilize equipment, instruments,
Autoclaves (3) and drying oven
and infectious waste
Microcentrifuge eppendorf, colony counters,
Incubator-provides a temperature-controlled
hot plate-stirrers, balances, pH-meter, fridges
environment to support growth of
PART 3: MEDIA PREPARATION
(MICROBILOGICAL PROCEDURE)
 SAMPLE
 CULTURE MEDIUM
- simple (peptone water and nutrient
agar)
-complex (blood agar - additional
ingredients)
-synthetic (defined, research purposes)
 GLASSWARE
FOOD SUPPLIED – the form of a mixture of
nutrient substances generally known as
medium.
THERE IS NO UNIVERSAL MEDIUM FOR THE
CULTIVATION OF ALL MICROORGANISM
BECAUSE DIFFERENT MICROOGRANISMS
REQUIRE DIFFERENT NUTRIENTS AND
ENVIRONMENTAL CONDITIONS.
MICROBIOLOGICAL CULTURE MEDIUM – a
substance the encourages the growth and
survival of microorganisms
THE CULTURE MEDIA MAY BE EITHER SOLID,
SEMI-LIQUID OR LIQUID based on
CONSISTENCY IN A FORM AND GENERALLY
CONTAIN NUTRIENTS, GROWTH PROMOTING
FACTORS, ENERGY SOURCES, BUFFER SALTS,
MINERALS, METALS, AND GELLING AGENTS.
TYPES OF CULTURE MEDIA (bacterial
culture media)
1. Liquid media – refereed as “broths”
- used in test-tubes, bottles, flask
- bacteria grow uniformly producing generally
turbidity
-aerobic bacteria grow as a thin film called
surface pellicle on the surface of undisturbed
broth
-used when a large number of bacteria have to
be grown (example: tryptic soy sauce)
- more associated with the examination of
food products
2. Social media – prepared by the addition of
solidifying agent
- agar or agar agar is the most commonly used
solidifying agent, it is not hydrolyzed by most
bacteria and doesn’t contribute any nutritive
property to the medium (1-3% concentration
used to make a solid agar medium) gelatin a
proteinaceous substance also used for gel-
forming with the concentration of 10-14% of
solution
- an unbranched polysaccharide obtained
from the cell membranes of some species of red
algae such as the genera gelidium
- more associated with the examination of
food products
COMMON SOLID MEDIA:
 Nutrient Agar
 Potato Dextrose Agar
 Malt Extract Agar
 Plate Count Agar
3. Semi- solid – it is formed when the amount
of agar is reduced to 0.2%- 0.5%
- fairly soft and used in demonstrating
bacterial motility and separating motile from
non-motile stains. (examples: STUARTS, AMIES
MEDIA, HUGH& LEIFSONS OXIDATION
FERMENTATION TEST)
BIOPHASIC MEDIUM – called when a culture
system comprises both liquid and solid medium
in the same bottle
CLASSIFICATIONS OF CULTURE MEDIA
1. Basal Media (generally media solid, liquid,
semi-solid)- simple media that support most
non fastidious bacteria (examples: PEPTONE
WATER, NUTRIENT BROTH and NUTRIENT
AGAR)
2. Enriched Media- additional nutrients in the
form of blood, serum, egg yolk and other
substances to provide more nutrients/ food for
organisms
3. Selective (agar based)/ Enrichment Media
(liquid in consistency) – formulated to inhibit
unwanted contaminating bacteria and help to
recover pathogen from a mixture of bacteria
4. Differential Media/ Indicator Media - are
substances used to recognize different bacteria
based on colony color (examples: MacConkey’s
agar, CLED agar and TCBS agar)
Preparation and Dispensing of Culture
Media
1.Clean, preferably pyrex glasses, must be used.
2. Dissolve ingredients in deionized or distilled
water.  MEDIUM
3.The pH value must be adjusted by using dilute
NaOH/HCl.
4. Microbiological culture media should not be
turbid and should contain no sediment.  Agar Stabs
Suspended matter can be removed by
centrifugation and/or filtration.  For filtration,
cotton wool or one to two sheets of folded filter
paper are usually sufficient.  
5. Much time and work can be saved by using
commercially produced media.  These are
available in powdered, granulated or liquid
forms.   sterilization, and the date of preparation must
Dispensing the medium  container.  It is a good practice to store media
The media are usually dispensed into:
a. test tubes of various sizes (Test tubes and
flasks are usually closed with cotton plugs or
metal caps)
b. screw-capped bottles. 
 If media are stored in cotton-plugged
vessels the plug should be covered with
aluminum foil or cellophane
 Contamination of the mouths of tubes
and flask must be avoided because it
leads to adhesion of the cotton plug to
the glass.
 Use of metal caps reduce the dust or
entry of other microorganism
 Caps should not be fit too tightly,
otherwise condensation may become
trapped in the vessel.
Preparation of Agar Slants/Slopes- prepared by
measuring approximately 5 ml of melted
medium into each tube, the still liquid medium
is allowed to solidify on a horizontal surface on
which glass rods or other supports are place so
as to raise the plugged ends of the tubes
Preparation of Agar Stab- prepared by
measuring approximately 5ml to 7ml of melted
medium into each test tube, liquid medium is
allowed to solidify on an upright position by
allowing to stand undisturbed on a test tube
rack.
AMOUNT AUTOCLAVING
CONTAINER
Broth 5-10ml Culture tube
Agar Slants 10ml Culture tube
7 ml Culture tube
Agar Plates 200 ml (20- 500 ml flask
25 ml per (2/3 full)
petri dish)
Storage of Culture Media
The composition of the medium, the method of
preparation, the pH, the conditions of
be indicated on the label of the medium
for several days at the temperature at which
they are eventually to be used.  At the end of
the incubation, any microbial contamination
may be identified.
 Media should be stored in a dust-free,
cold (about 4 °C), dark (or protected
from direct sunlight) and not too humid
room.  
 Screw-capped bottles are suitable for
storing media.  They have aluminum
caps or plastic caps, both of which
 withstand autoclaving.  Screw-capped
bottles can be tightly closed.  sterilized and place them in the dry
 Media stored for periods longer than 6
months should be checked before use.
TO PREPARE A CULTURE MEDIUM THERE IS
A COMPUTATION
EXAMPLE: If the label of the bottle directs
you to add 28 grams of medium to 1000 mL
of distilled water, how many grams of
powdered medium should you weigh to
make 200 mL?
28 g Xg
SOLUTIONS: =
1000 g 200 g
1000 x = 28 x 200
5600
X=
1000
X= 5.6 grams
PART 4: STERILIZATION
Sterility- absence of any kind of living organism.
METHODS OF STERILIZATION
1. Sterilization by Dry Heat (conventional oven)
- glassware, petri dish and pipette are sterilized
in the dry state in the hot air oven, exposed to
160 to 165 C for 2hrs or 170 to 180 C for 1hr.
The period of heating commences from the
time the oven temperature reaches 160 °C.
Heating to redness.  Wire needles and loops
used for inoculation can be sterilized by heating
the wire to redness.  The straight wire and the
loop should be held obliquely in the upper part
of the Bunsen flame, both before and after use,
until they begin to glow.   UV light is very low, this kind of radiation is
Flaming.  When glass vessels, test tubes or
flasks are open, their mouths and metal caps
should be flamed to prevent microbial
contamination of cultures.  Scalpels, spatulas
and glass rods should be immersed in alcohol
before flaming.
PROCEDURES IN DRY STERILIZATION
1. Prepare the glassware to be
air oven.
2. Set the thermostat at 170 °C and
sterilize the materials for one hour.
2. Sterilization by Moist Heat (autoclave) -
Culture media, solutions and cotton are usually
sterilized by this method. The shortest time for
effective sterilization is that required for
destruction of the most heat-resistant
microorganisms.  At an approximately neutral
pH, bacterial spores are the most resistant.
Steam under pressure at varying temperature-
and-time intervals, depending on the materials
sterilized, is used.  A temperature of 121 °C for
15 lbs steam per square inch, for 15 minutes is
used for most media.
3. Fractional Sterilization in Flowing Steam
Tyndallization, or intermittent sterilization, is
used when steam pressure heating is too
rigorous.  The material to be sterilized is
exposed to short periods of steaming at
intervals of 12 to 14 hours.  Between steaming,
the media is often placed at incubator
temperatures.
4. Filtration -Microbial cells can be removed
from fluids and gases by filtration.  In industrial
microbiological processes, heat-sensitive fluids
as well as the laboratory air are sterilized by
filtration.
- Large volumes of air, such as are required in
clean rooms and laminar-flow cabinets, are
sterilized by filters.  Dryness is a pre-requisite of
the effectiveness of any air filter.
5. Radiation (UV) - the penetration capacity of
effective only for the sterilization of clean
rooms and laboratory surfaces. Radiation of
wavelength 253 to 265 nm is the most
effective.  However, care must be taken to
protect the skin and eyes.   
6. Cleaning and Disinfection
Cleaning.  It is important to remove surface-
active substances by thorough cleaning and
rinsing before sterilization.  For glassware,
soaking in detergent and washing with a brush
is the most applicable method
- Cleaned glassware and instruments should
be rinsed with tap water followed by
several rinses with distilled water, and
after draining, dried in a drying cabinet.
Disinfectants- used to reduce the numbers of
microorganisms on surfaces.  Ethanol is widely
used as a disinfectant for metal instruments,
working surfaces, hands, rubber tubes and
rubber stoppers.  It is most effective as an
aqueous solution of about 70%.  beneath the surface of the medium when it
- Other disinfectants are calcium
hypochlorite or sodium hypochlorite,
iodophores, formaldehyde and
quarternary ammonium compounds.  A
2% solution of hypochlorite contains 0.5%
active chlorine.  Iodophores are aqueous
solutions of iodine and non-ionic surface-
active compounds.  Formaldehyde is used
in 1% to 5% solutions.  Quarternary
ammonium compounds are used in the
form of warm solutions.
PART 5: CULTURAL
CHARACTERISTCS OF
MICROORGANISM
Culture- when bacteria or other microorganism
grow in laboratory medium. Different species of
bacteria growing on the same kind of medium
may appear different.
Microorganisms exist in nature as a
mixed culture.  However, to determine
the characteristics of a particular
species, it is important that the
organism be isolated and grown in the
laboratory as a pure culture (a culture
containing only one species of
organism).
TECHNIQUES OF ISOLATING PURE CULTURE
1. Streak Plate or Spread Plate Technique.  The
streak plate or spread plate is obtained by
transferring a portion (usually 0.1 mL) of a
bacterial suspension on the surface of an agar
medium and streaking or spreading over the
surface.  Spread plates are usually streaked with
a sterile bent glass rod.  This process thins out
the bacteria on the agar surface so that
individual bacteria are separated from each
other.
2. Pour Plate Technique- involves the principle
of dilution or thinning out the concentration of
the bacterial suspension.  Agar medium is
poured into the culture plate, allowed to solidify
and incubate.  Some organisms may be trapped
gels.  With this, the pour plate exhibits both
surface and sub-surface colonies.
CHARACTERISTICS OF COLONY
TRANSFORMATION
 Agar plate cultures are obtained by the
pour plate technique or the spread
plate technique. 
 Agar slant cultures are prepared by
making a streak inoculation up the
middle of the sloped surface with a
transfer needle. 
 For the stab culture, the transfer needle
carrying the inoculum is introduced in a
straight line from the top to the bottom
of the tube and removed along the
same path. 
 Tubes of broth can be inoculated with
the transfer needle or loop.
Agar Plate Colonies
Size.  Colonies range in size from extremely
small (pinpoint) measuring only a fraction of a
millimeter in diameter, to large colonies
measuring 5 to 10 mm in diameter.
Margin or Edge.  The periphery of bacterial
colonies makes many different patterns,
depending upon the species.  It may be very
evenly circular, like the edge of a droplet, or it
may show such irregularities as rounded
projections, irregular notches, threadlike
projections or root-like projections.
Elevation.  The colonies may be very thin (flat)
to thick (raised).  Raised colonies exhibit varying
degrees of a convex structure.
Chromogenesis.  Colonies may be colored
(pigmented) or not (nonpigmented).  Various
shades of red, yellow, brown and violet
characterized certain species. Growth in Nutrient Broth
Optical features.  May be opaque, translucent,
or opalescent. Amount of Growth.  Scanty, moderate or
abundant.
Distribution of growth throughout the
broth. Uniformly distributed (evenly turbid); 
growth confined to surface of broth as a scum
or film (pellicle);  growth accumulated as
sediment, which may be granular or viscous.
Odor.  May be putrid, fruity or aromatic, or
negligible.

Agar Slant Growth

Amount.  Scanty, moderate or abundant.
Margin or Edge of Growth.  Uniform or even or
exhibiting various irregularities similar to the
irregularities described or colonies.
Consistency of mass of growth.  Butyrous or
butter-like consistency; viscous or stringy; dry
and brittle.
Chromogenesis.  Similar to that described for
colonies.
Growth in Agar Stabs
Amount of Growth.  Scanty, moderate or
abundant.
Margin or Edge of Growth.  Uniform or even or
exhibiting various irregularities similar to the
irregularities described or colonies.
Chromogenesis.  Similar to that described for
colonies
 Aspergillus flavus

penicillium

Alternaria

Botrytis Cinerea

YEAST COLONIES - Young colonies are

ointment-like in consistency, while older ones
are denser, tending to dry out, and may form
pigments which give the organism a
characteristic color (yellow, orange, pink, brown
or black).  The appearance of yeasts in liquid
culture may also be used to identify the
organism.  Some species grow on the bottom of
the culture vessel forming a sediment, whereas
other species exhibit a uniform turbidity. 
Another group grows only at the surface,
forming a thicker layer.
MOLD COLONIES-The presence of molds in food
is characterized by the fuzzy, cottony
appearance that tend to scatter on the surface
as it grows. described based on the color of the
mycelium and the extent at which the organism
spread in the medium.  is the micrometer, which is equivalent to
NAME IMAGE
Mold culture
Rhizopus Stolonifer
Geotricum candidum
PART 6: MORPHOLOGY OF
BACTERIA
Morphological Characteristics
 Size of the cells - is determined by using
a microscope equipped with an ocular
micrometer (a disk with etched,
equidistant lines, the distance between
them pre-determined by reference to a
stage micrometer. 
- The unit for bacterial measurement
1/1000 mm or 1/25,400 inch.
 the shape
 arrangement of the cells in groups
 the differentiation of their fine
structures. 
 3 General forms of bacterial cells
1. Spherical or ellipsoidal- occur singly, in
pairs (diplococcus), in chains
(streptococcus), in irregular grapelike
clusters (staphylococcus), as group of cocci
in a quadratic arrangement (tetrads), or as
package-like cubes of eight cocci (sarcina).
Arrangement of the round-shaped
bacteria
2. Cylindrical or rod-shaped- [bacillus; bacilli
(pl)] do not arrange themselves in a variety of
patters similar to the cocci.  They often appear
as single cells, but many cells occur in pairs
(diplobacillus) or form chains (streptobacillus).
3. Spiral or helical- [spirillum; spirilla (pl)] occur
predominantly as individual cells.
Preparation of Smears and Simple
Staining  
Aniline dyes- most stains used in microbiology
derived from benzene.
- usually salts, although a few are acids or
bases, composed of charged colored ions. 
Chromophore – ion that is colored.
Methylene blue chloride Methylene
blue   +   Cl−
+
(chromophore)
If the chromophore is a positive ion, like
methylene blue, the stain is considered a basic
stain; if a negative ion, it is an acidic stain. 
Most bacteria are stained when a basic stain
permeates the cell wall and adheres by weak
ionic bonds to the negative charges of the
bacterial cell.
Simple Stains- procedure that use only one
stain. (Methylene blue, crystal violet, malachite
green, basic fuschsin carbolfuschin and
safranin)
- Simple stains can be used to determine cell
morphology, size and arrangement.
 Direct Stain- a simple stain that
colors the bacteria
 Negative Stain- colors the
background but leaves the bacteria
 unstained. (eosine acid fuchsin, in the cell to form a crystal violet-iodine
rose Bengal and congo red)  complex (CV – I).
 Smear is made by spreading a bacterial 3. Apply decolorizing agent (ethyl alcohol
suspension on a clean slide and allowing it to or ethyl alcohol-acetone).  The primary
air-dry.  stain is washed out (decolorized) of
- The dry smear is passed through a Bunsen some bacteria, while others are
burner flame several times to heat fix the unaffected.
bacteria.  4. Apply secondary stain  or counterstain
- Heat fixing denatures bacterial enzymes, (safranin).  This basic dye stains the
preventing them from digesting cell parts, decolorized bacteria red.
which causes the cell o break, a process
known as autolysis. 

Gram- negative- decolorize

- The alcohol dissolves the outer
lipopolysaccharide layer and the CV-I
washes out through the thin layer of
peptidoglycan

 Gram- positive- those that retain the primary

stain
- cell walls consist of many layers of
peptidoglycan.
-   The CV-I is larger than the crystal violet or
iodine molecules that initially entered the
cell and cannot pass through the thick
peptidoglycan.  In gram-negative cells, the
alcohol dissolves the outer
lipopolysaccharide layer, and the CV-I
washes out through the thin layer of pept

The Gram Stain  

Gram stain - allows you to classify bacteria as

either gram-positive or gram-negative.
- useful stain for identifying and classifying
bacteria. 

The staining technique consists of the

following steps:

1. Apply primary stain (crystal violet).  All

bacteria are stained purple by this basic
dye.
2. Apply mordant (Gram’s iodine).  The
iodine combines with the crystal violet RESULT OF GRAM STAINING