Menofia University

Faculty of Science
Botany and Microbiology Department

Practical
General Microbiology

Prepared by

Prof. Dr. Sabha El-Sabbagh

2021/2022
 :‫رسالة الكلية‬

‫تقدم كلية العلوم برامج دراسية و بحثية ذات طابع أكاديمي و تطبيقي‬
‫طبقا للمعايير األكاديمية المرجعية و المعايير األخالقية و تساهم فى‬
‫خدمة المجتمع و تنمية البيئة‬
The faculty of science offers academic
programs and applied research . compatible
with the national academic reference
standards , ethical standards and contributes to
the service of its community
:‫رؤية الكلية‬
‫تسعى كلية العلوم ان تكون متميزة فى العلوم األساسية و قادرة على المنافسة محليا‬
‫و أقليميا‬
To be distinguished in the basic science and able to
compete locally and nationaly
 Practical Microbiology

Student Name …………..………………………………………

Branch …………..………………………………………

Level …………..……………………………………….

Section …………..………………………………………

Term/Year Second Term (2021/ 2022)

2022/2022
 Contents

No Subject Page
1 MICROSCOPY 1
2 RESOURCES FOR PRACTICAL 4
MICROBIOLOGY
3 MEDIA, STERILIZATION AND 7
DISINFECTANTS
4 MICROORGANISMS
4. 1. Bacteria 12
4. 2. Cyanobacteria 18
4. 3. Protista 19
4 3. 1. Algae 22
4. 3. 2. Fungi 37
5 49
Microbial Growth Oxygen and the Growth of Bacteria
55
Other Influences On Microbial:Growth: Osmotic
Pressure and pH

Questions 60
REFERENCES 64

Intended Learning outcomes (ILOs)

By the end of this course the student should be able to:
1- Recognize the different methods for sterilization and disinfection
techniques.
2- Differentiate between categories of microorganisms.
3- Microscopically, identify the most common forms from each category.
4- Describe the features of representative forms from each category of
microorganisms.
1- MICROSCOPY
1. 1. Using the microscope

1- Turn on the microscope and check that the light is working.

2- Place a piece of thin tissue on a slide with a coverslip on top. 3- Make sure the
lowest power objective is in place.
4- Adjust the eyepieces to a comfortable viewing position.
5- Focus by lowering the objective towards the slide while watching from the side.
Then look through the microscope and focus up slowly until the tissue comes into
focus. Never focus down towards the slide.
6- Close the condenser so that the rim is just visible through the microscope and
focus finely on it.
7- The condenser rim should be centered in the field of view. If it is not, refer to the
microscope manufacturer's handbook to findout how to centre it.
8- Now change to the next higher power objective and repeat the focusing of the
objective and condenser.
9- You should always view specimens with the condenser just outside the field of
view. Adjust light levels with the light power switch.
10- Take your time adjusting the microscope to get it just how you want it. You will
probably need to readjust it whenever someone else uses it.

1. 2. Light Microscope

Light microscopes are fairly simple, consisting of a sturdy base, an adjustable

arm, a stage for the specimen, a focusing knob, an eyepiece lens and an objective
lens. Some have electrical light sources, while inexpensive models make use of a
mirror and available room light.

1
Lab1. NOTES for using a microscope
1- Always carry the microscope with one hand on the Arm andone hand on the
Base. Carry it close to your body.
2. Remove the cover, plug the microscope in.
3. Always start and end with Low Power.
4- If you wear glasses, take them off; if you see only your eyelashes, move closer.
Be sure to close, or cover your other eye!!
NOTE: If you see a dark line that goes part way across the field of view, try
turning the eyepiece. That dark line is a pointer that will be very valuable when
you want to point out something to your lab partner, or your demonstrator.
5. If, and ONLY if, you are on LOW POWER, lower the objective lens to the lowest
point, then focus using first the coarse knob, then the fine focus knob. The
specimen will be in focus when the LOW POWER objective is close to the
lowest point, so start there and focus by slowly raising the lens. If you can’t
get it at all into focus using the coarse knob, then switch to the fine focus knob.
6. Adjust the Diaphragm as you look through the Eyepiece, and you will see that
MORE detail is visible when you allow in LESS light! Too much light will give the
specimen a washed-out appearance.
7. Once you have found the specimen on Low Power (100x), unless specifically
asked to draw it on low power, center the specimen in your field of view, then,
without changing the focus knobs, switch it to High Power. If you don’t center
the specimen you will lose it when you switch to High Power (Yellow).
8. Once you have it on High Power remember that you only use the fine focus
knob! “Caution, do not remove the slide when it is on High Power.” -- The
High Power Objective (400x) is very close to the slide. Use of the coarse focus
knobwill scratch the lens, and crack the slide.
9. NEVER USE THE OIL LENS. It is an oil immersion lens. Without the oil to
lubricate the lens, you will destroy it . Also, the oil is needed to help gather
enough light to actually see through the lens.

2
Q1: Try to label the different parts of the microscope in the proper
place.

1 9
2 10
3 11
4 12
5 13
6 14
7 15
8 16

3
Lab 2- Resources of practical microbiology
2. 1. Equipments

Equipment Use
Loop Routine inoculation of agar slopes/deeps and
(wire/plastic) small volumes of liquid media (up to ca 10cm3);
making streak plates.
Spreader Spreader (glass/plastic) Making lawn/spread
(glass/plastic) plates
Forceps Transfer of sterile paper/antibiotic discs; also
(metal/plastic) plant material, e.g. short lengths of root with
nodules
Pipette Transfer of measured volumes/drops of
(calibrated/ culture/sterile solutions (dry, non-absorbent
dropping; cotton wool plug in neck prevents
glass/ plastic) contamination)
Teat Filling and emptying pipettes safely (never
pipette by mouth)
Test tube Small volumes (ca 5-10 cm3) of liquid
media/agar slopes/sterile solutions for
inoculation (held in test tube rack; dry non-
absorbent cotton wool plug or plastic cap
prevents contamination)
Conical flask Large volumes of liquid media for inoculation
and liquid/media for short-term storage
(nonabsorbent cotton wool plug prevents
contamination but does not reduce evaporation
during long storage)
Petri dish Plastic: pre-sterilized for streak/spread/lawn/
(plastic/ glass) pour plates; Glass: only for materials for
sterilization by hot air oven, e.g. paper discs
Marker pen Labeling Petri dishes, test tubes, flasks, bottles
and microscope slides
Personal Clean laboratory coat/apron: protection of
protective clothing, containment of dust on clothing; Safety
equipment spectacles: not considered essential when
dealing with suitable cultures and chemicals

4
 2. 2. Apparatus
Apparatus Use
Bunsen burner Sterilization of wire loops and (with alcohol)
metal forceps and glass spreaders.
Impervious sheet or Provides individual student working area if the
tray bench surface is not appropriately sealed.
Autoclave/pressure Sterilization of media, solutions and
cooker equipment before use and contaminated items
afterwards; melting solidified agar media for
use.
Gas ring/hot plate Steam generation in autoclave
Autoclavable/roasti Holds contaminated items in autoclave to
ng bag contain spillages
Hot air oven Sterilization of glass Petri dishes and pipettes
and paper discs (but not essential as
autoclaves/ pressure cookers serve virtually
all needs)
Discard pots Disposal of used pipettes and slides of non-
containing stained microscopical preparations
disinfectant
Microwave oven Melting solidified agar media for use (but not
in vessels with metal caps and not for
sterilization)
Incubator Incubation of cultures (but many cultures will
grow at room temperature in the interval
between lessons)
Water bath Suitable temperature for keeping melted agar
media molten for use (ca 50ºC); accurate
temperature control.
Thermometer Checking incubator/water bath temperatures.
pH meter Checking and adjusting pH values of media.
Cupboard Storage of culture media and stock cultures.
Refrigerator Storage of heat-labile materials.
Microscope, slides, Microscopical observations
cover slips, stains,
staining
rack, immersion oil

5
Q2: What are the main purposes for using the following:

a- The incubators:
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- The Autoclave ………………………………………………………………
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D- Wire loop: …………………………………………………………………..
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E: Disinfectants: ………………………………………………………………
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6
Lab 3 Sterilization and disinfection
Media preparation
There are different media which support the microbial growth; each
medium has definite composition that favor the microbial requirements for growth.
Physically, there are three kinds of media; liquid, solid and semi solid media. The
differences among them is the addition of solidifying agent (agar) to get the liquid
medium into solid medium. For preparation of certain medium the ingredients are
weighted and dissolved in definite volume of distilled water, dissolved and sterilized
by autoclaving.

Sterilization means the complete destruction of all the micro- organisms including
spores, from an object or environment. It is usually achieved by heat or filtration but
chemicals or radiation can be used.
Disinfection is the destruction, inhibition or removal of microbes that may cause
disease or other problems e.g. spoilage. It is usually achieved by the use of
chemicals.

A- STERILIZATION

Use of the autoclave/pressure cooker

The principle of sterilization in an autoclave or pressure cooker is that steam under

pressure is used to produce a temperature of 121ºC which if held for 15 minutes will
kill all micro-organisms including bacterial endospores.

Sterilization of equipment and materials

a- Wire loop
Heat to redness in Bunsen burner flame.

b- Empty glassware and glass (not plastic!) pipettes and Petri dishes

7
 Either, hot air oven, wrapped in either greaseproof paper oraluminium and
held at 160ºC for 2 hours, allowing additional time for items to come to
temperature (and cool down!).Or, autoclave/pressure cooker.
Note: plastic Petri dishes are supplied in already sterilizedpacks, they are
disposable.

Oven temperatures and time for sterilization120°C for 8 hours

140°C for 3 hours160°C for 1 hour
180°C for 20 min.

c- Culture media and solutions

Autoclave/pressure cooker.
Autoclave pressures and temperatures:
5 psi at 107°C 7 psi at 110°C 10
psi at 115°C 15 psi at 121°C 20
psi at 126°C

d- Glass spreaders and metal forceps

Flaming in alcohol (70%).

e- Other methods of sterilization are:

1 Ultraviolet radiation: can be useful for benches and clean rooms.
2 Gamma radiation: is used for the sterilization of plastics in industry.
3 Filtration: using filters of a maximum pore size of 1nm, generally used to sterilize
small quantities of liquids which are unstable at high temperatures.

10
Q3: Complete the following:
a- Agar is used to prepare...................................... media.
b- The stored agar media could be re-molen by
…………………….., or ......................................... , or
……………………………………………………………………c- For storing the
Petri dishes, the media containing base
should be uppermost this is to
……………………………………………
d- Agar slants are prepared by pouring agar media in
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e-
Q4- What are the differences between sterilization anddisinfection?
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name of the equipment used to sterilize media and liquids?
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Q6: What are the physical methods for sterilization?
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Q7: What is the mechanical method for sterilization?
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11
Lab 4- Bacteria; shapes and motility
Bacteria possess 4 main forms which are spherical (cocci), rod-shaped (Bacilli),
spiral-shaped (vibrions and spirilla), and filamentous (actinomycetes).

(a) Coccus (Pl. Cocci): These are spherical cells which existsingly or in groups
of two or more cells.
(b) Bacillus (Pl. bacilli): These are small rods which may beflagellated or non-
flagellated.
(c) Spiral-shaped
(d) Filamentous Motility in

Bacteria

Bacteria are either non-motile or motile. The motile forms are either creeping or
swimming. Creeping bacteria move or creep slowly on a supporting surface as a
result of wave-like contractions of their bodies. Swimming bacteria move freely in a
liquid medium due to presence of flagella.

1 Monotrichous (one flagellum attached to one pole of thecell).

2 Lophotrichous (A tuft of flagella at one pole of the cell).
3 Amphitrichous (A single or tuft of flagella at the two polesof the cell).
4 Peritrichous (Many flagella distributed over the wholesuface of the cell).

12
13
14
Q8: Examine the specimens of the mounted bacteria, draw thecells and try to
describe their shapes and characters.
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provided specimens, describe and draw a diagram for the motile bacteria.
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15
Lab 5- Gram Staining of bacterial cells
A “smear” of bacteria or yeast is made on a microscope slide, fixed, stained, dried
and, without using a coverslip, examined with the aid of a microsope. Aseptic
technique must be observed when taking samples of a culture for making a smear. A
culture on agar medium is much preferable to a liquid culture for making a smear.
A smear that
is thin and even enables the shape and arrangement of cells to be clearly seen and
ensures that the staining procedure is applied uniformly.
1. Put the slide with the fixed smear uppermost on a staining rack over a sink or
staining tray.
2. Thoroughly cover the smear with crystal violet solution and
leave for 1 minute.
3. Hold the slide with forceps (optional but avoids stained fingers), at a 45° angle
over the sink.
4. Pour off the stain, wash off any that remains (and any on the back of the slide)
with iodine solution.
5. Put the slide back on staining rack.
6. Cover the smear with iodine solution and leave for 1 minute. Iodine solution acts
as a “mordant” (a component of a staining procedure that helps the stain to
adhere to the specimen), a crystal violet-iodine complex is formed and the smear
looks black.
7. Hold the slide with forceps at a 45° angle over the sink wash off the iodine solution
with 95% (v/v) ethanol (not water); continue treating with alcohol until the
washings are pale violet.
8. Rinse immediately with tap water.
9. Put the slide back on staining rack.
10. Cover the smear with the counter stain, e.g. safranin solution, 0.5% w/v, for 30
seconds.
11. Rinse off the stain with tap water.
12. Blot dry the smear with filter/fibre free blotting paper using firm pressure but not
sideways movements that might remove the smear.
13. Examine under oil immersion.
14. When finished, dispose of slides into discard jar.

16
Q5: How can you make a smear for a bacterial culture?
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Q2: What are the main steps for Gram's staining?

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Q3. What are the colours of Gram+ve and Gram-ve bacterialcells?

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Q4. Mention the reason for the differences in colour of G-ve andG+ve stained
bacterial cells.
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17
Lab 6- Cyanobacteria
Cyanobacteria are prokaryotes (single-celled organisms) often referred to as "blue-
green algae." While most algae is eukaryotic (multi-celled), cyanobacteria is the only
exception. Cyanobacteria obtain their energy through photosynthesis. They are not
strictly unicellular, but can be found in colonial and filamentous forms, some of
which differentiate into varying roles. For example, cyanobacteria specialized for
nitrogen fixation are called heterocysts

Nostoc
Nostoc is a diverse genus of Cyanobacteria. They are found in gelatinous colonies,
composed of filaments called "trichomes" surrounded by a thin sheath.

18
Q6: Examine the provide specimens for Nostoc, draw a labeled diagram to the
filament with its different parts, and describe itsimportant features.
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19
Lab 7- Protozoa
Protists are microscopic unicellular organisms that don't fit into the other kingdoms.
Some protists are considered plant-like while others are considered animal-like. The
animal-like protists are known as protozoans. The amoeba is considered an
animal-like protist because it moves and consumes its food. Protists are classified
by how they move, some have cilia or flagella, but the amoeba has an unusual
way of creeping along by stretching its cytoplasm into fingerlike extensions called
pseudopodia. The word "pseudopodia" means "false foot". Label the
pseudopodia. When looking at amoeba under a microscope, an observer will note
that no amoebas looks the same as any other, the cell membrane is very flexible
and allows for the amoeba to change shape. Color and label the cell membrane
red. Amoebas live in ponds or puddles, and can even live inside people.

20
Q7: Examine the provide specimens for Amoeba, draw a labeleddiagram, and
describe the important characters of the cell.

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21
Lab 8 - Algae; Phylum: Euglenophyta
Euglena

The many species of Euglena and some of the related organisms,all called
flagellates, form an interesting group of unicellular creatures since, although the
majority are plant-like in their feeding, a few feed saprophytically (like fungi), some
holozoically (like animals), and nearly all of them possess characteristics which
make them more like animals than plants.

22
Q8: Examine the provided specimen with the help of microscope and draw a labeled
diagram to Euglena and describe its cellular structure and the distinguished cellular
components.

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23
Lab 9- Algae; Phylum: Chlorophyta
a- Chlamydomonas

Chlamydomonas is the name given to a genus of microscopic, unicellular green

plants (algae) which live in fresh water. Typically their single-cell body is
approximately spherical, about 0.02 mm across, with a cell wall surrounding the
cytoplasm and a central nucleus. Two filaments of cytoplasm, flagella, (sing.
flagellum), extend from one end, and their whip-like lashings pull the
Chlamydomonas through the water and rotate it at the same time. A single, cup-
shaped chloroplast occupies the greater part of the cell. In this chloroplast is a
protein region called a pyrenoid, which is involved in starch production and may be
surrounded by starch granules.

24
Q9-1: With the help of the microscope draw a labeled diagram to Chlamydomonas
and describe its cellular structure and the function of cellular components.

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25
Phylum: Chlorophyta
b- Pandorina

26
Q9-2: With the help of the microscope draw a labeled diagram to
Pandorina, and describe its colonial structure.

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Phylm: Chlorophyta

c- Volvox

28
Q9-3: With the help of the microscope draw a labeled diagram to Volvox, and
describe its colonial structure and the function of the colonial cells components.

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29
Phylum: Chlorophyta

d- Spirogyra

Spirogyra is a filamentous alga belongs to chlorophyta, The cell is cylindrical

elongated. It has a distinguished dentated spiral ribbon-shaped chloroplast with
pyrenoids spread along the chloroplast. The cytoplasm is lining carrying the
nucleous in the center. The cell wall is cellulosic.

Spirogyra cell (3D)

30
 Spirogyra conjugation
At certain times of the year, tubular structures grow out from each cell of a pair of
filaments lying parallel to each other. The tubes join up to make a passage between
each cell and its partner. The chloroplasts and other structures become less distinct
and the cytoplasm pulls free from the cell wall to form a rounded structure. The
cytoplasmic contents of the cells of one filament then pass through the tube
(conjugation tube) and fuse with the cytoplasm of the cells of the adjacent filament.

31
Q9-4 - With the help of the microscope draw a labeled diagram toSpirogyra
vegetative filament, and describe the cellular structure and function.

Q: 2- With the help of the microscope draw a labeled diagram toSpirogyra conjugative
filaments, and describe the steps of reproduction.

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32
Lab 10- Algae; Phylum: Bacillariophyta
(Diatoms)

Diatoms have their cells surrounded by a silica-based shell known as a frustule,

composed of two overlapping halves (the epitheca and the hypotheca). From the
side view of the frustule we can see the girdle that hold these two haves together.
According to the silica striations on the cell wall there are two kinds of cells, centrally
(radially) and bilaterally symmetrical deposition. In bilaterally symmetrical (long, thin)
diatoms, meiosis in parental cells produces identical, non-motile gametes, which
fuse to form a zygote.

33
Q10-1 - With the help of the microscope draw a labeleddiagram to diatoms
cells, and describe the cellular
Q10-2- What are the different views of diatoms, and the forms of silicate striations
in diatoms?

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34
Phylum: Phaeophyta (Brown algae)

Fucus

The thallus is perennial with an irregular or disc-shaped holdfast. The erect portion
of the thallus is dichotomous or subpinnately branched, flattened and with a distinct
midrib. The gametangia develop in conceptacles embedded in receptacles in the
apices ofthe final branches. They may be monoecious or dioecious.

Q:10-3- With the help of the microscope draw a labeled diagram

35
 to Fucus cells, and mention the general characters of thealgal structure.

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Q2: Examine the provided specimens for male and female conceptacles of Fucus and
draw a labeled diagram to each.

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36
Lab 11- True fungi Phylum:
ZygomycotaRhizopus
Hyphae of Rhizopus are coenocytic, but few dividing walls or septa. A sporangium
is a structure inside which spores are developed. It is held a lot on an aerial hypha
called a sporangophore. A thick protective covering develops around the dikaryon in
Rhizopus, forming the zygospore, which can survive extremes of draught and
temperature and may remain dormant for months. When conditions are favourable
again.

2 3 4
1 5

37
Q:11-1 Examine the provided specimens of mould bread (Rhizopus) and prepare a
slide for microscopic examination.
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Q11-2: Examine the provided specimens of Rhizopus, and draw alabeled diagram to
the vegetative and sexual stages.

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38
Phylum: Ascomycotina

Yeasts (Saccharomyces cerevisiae)

Yeasts are unicellular fungi reproduce by budding, although some species with yeast
forms may become multicellular through the formation of a string of connected
budding cells known as pseudohyphae, or false hyphae, as seen in most molds.
Yeast size can vary greatly depending on the species, typically measuring 3–4 µm
in diameter, although some yeasts can reach over 40 µm.

39
Q12c Prepare a slide from the provided baker's yeast(Saccharomyces)
and examine its structure microscopically.

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Q12c: Draw a labeled diagram to the vegetative yeast cells, anddescribe the
cellular feature and structure.

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40
Lab 12- Fungi; Phylum: Ascomycotina
Aspergillus
The mycelium of Aspergillus are well developed, richly branched and septated.
Asexual reproduction: Is the most common type of reproduction. It takes place
through the production of conidia. Conidiophore arises as aerial unbranched hypha
from the mycelia. The cell from which the conidiophore emerges is known as foot
cell. Conidiophores are produced singly and are non- septate unbranched and each
ends with a head known as vesicle. From the vesicle arises a large number of
phialides which are either in one or two series.

41
Q12: Examine the provided specimen on which Aspergillus is grown, and write
the important features of its colony.

Q12-2: Mount a slide with the grown fungus Aspergillus, and drawa labeled diagram
to it.

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Phylum: Ascomycotina

Penicillium

The mycelium is more or less similar to that of Aspergillus, conidiophore is septate

branched and ends with cluster of phialides giving the characteristic brush-shape of
Penicillium(small brush). Conidial formation and sexual reproduction are similar to
those of Aspergillus

43
Q13b: Examine the provided specimen on which Penicillium is grown, and write
the important features of its colony.

Q13b: Mount a slide with the grown fungus and draw a labeled diagram to it.

Q13b: Compare between Aspergillus and Penicillium.

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Phylum Ascomycotina

Peziza

Peziza, is an ascocarpic fungus. The cup-shaped fruiting bodies are colored on the
interior surface. It consists of a palisade layer composed from asci and
paraphysis. Each ascus contains 8 ascospores and the paraphyes represent the
sterile hyphae thatdevoid of ascospores.

45
Q13-1: Examine the provided fruit body (Ascocarp) of Peziza and describe its
morphology.

Q13-2: Examine the provided slide of T. S. in Peziza Ascocarp, draw a labeled

diagram to the section of Peziza fruit body and its components.
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Phylum Basidiomycotina
Mushroom (Agaricus)
The mature fructification sporophore is an umbrella-like structure consisting of a
stout and rigid stalk (stipe) which bears a round cap (pileus). From the cap the gills
hang down vertically and the number of gills in one cap ranges from 300 to 600. The
stipe consists of two layers of interwoven hyphae which makes
pseudoparenchymatous tissue. The outer layer called the cortex, whereas the inner
or the central region is made up of loosely arranged hyphae having air spaces. The
gill region is made up of central portion of interwoven hyphae called the trama.
Around the trama a compact zone of hyphae is found forming the subhymenium.
An outer surface layer called hymenium which composed of basidia and
paraphysis. The basidium is non- septate and club-shaped. Each basidium carries 4
basidiospores on short sterigmata.

47
Q13-3: Examine the provided specimen of Mushroom fruit body, draw a labeled
diagram to the whole fungus with its different parts.

Q13-4: Examine the provided slide mounted with T.S of the mushroom gill,,
draw a labeled diagram of the internal structure of the gill with the
basidiospores.

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48
 Microbial Growth

Oxygen and the Growth of Bacteria

Objectives
After completing this exercise you should be able to,

1. Identify the incubation conditions for each of the Following types of

organisms: aerobes, obligate an aerobes, aerotolerant
anaerobes,microaerophites. and facultative anaerobes.

2. Describe three methods of culturing anaerobes.

3. Cultivate anaerobic bacteria

Background
The presence or absence of molecular oxygen (O2) can be very
important to the growth of bacteria. Some bacteria, called obligate
aerobic bacteria, require oxygen, while others, called anaerobic bacteria,
do not use oxygen.

One reason obligate anaerobes cannot tolerate the presence of oxygen

is that they lack catalase, and the resultant accumulation of hydrogen
peroxide' is lethal. Aerotolerant anaerobes cannot use oxygen but
tolerate it fairly well. although their growth may be enhanced by
microaerophiles conditions. Most of these bacteria use fermentative
metabolism.

Some bacteria, the microaerophiles, grow best in an atmosphere with

increased carbon dioxide (7% to 10%) and lower concentrations of

49
 oxygen. Microaerophiles will grow in a solid nutrient medium at a depth
to which small amounts of oxygen have diffused into the medium (Figure
).

In order to culture microaerophiles on Petri plates and nonreducing

media a candle jar is used. Inoculated plates and tubes are placed in a
large jar with a lighted candle. After the lid is placed on the Jar, the
candle will extinguish when the concentration of oxygen decreases (the
concentration of carbon dioxide will be raised).

The majority of bacteria are capable of living with or without

oxygen; these bacteria are called facultative anaerobes (Figure ).

Four genera of bacteria lacking catalase are Streptococcus

Leuconostoc, Lactobacillus, and Clostridium Species of Clostridium are
obligate anaerobes, but the other three are aerotolerant anaerobes. The
three aerorolerant anaerobes lack the cytochrome system to produce
hydrogen peroxide and therefore do not need catalase Determining the
presence or absence of catalase can be very helpful in identifying
bacteria. When a few drops of 3% hydrogen peroxide are added to a
microbial colony and catalase is present, molecular oxygen is released
as bubbles.

In the laboratory, we can culture anaerobes either by excluding

free oxygen from the environment or by using reducing media. Many
anaerobic culture methods involve both processes. Some of the
anaerobic culturing methods are as follows:

1-Reducing media contain reagents that chemically combine with free oxygen,
reducing the concentration of oxygen. In thiogiycollate broth sodium
thiogiycollate (HSCH2 COONa) will combine with oxygen. A small amount

50
 of agar is added to increase the viscosity, and this reduces the diffusion of air
into the medium. Usually dye is added to indicate where oxygen is present in
the medium. Resazurin, which is pink in the presence of excess oxygen and
colorless when reduced, or methylene blue are commonly used indicators.

2-Conventional, non reducing media can be incubated in an anaerobic

environment. Oxygen is excluded from a Brewer anaerobic jar by
adding a Gas-Pak(R) and a palladium catalyst to catalytically combine
the hydrogen with oxygen and form water (Figure ). Carbon dioxide
and hydrogen are given off when water is added to the Gas-Pak(R)
envelope of sodium bicarbonate and sodium borohydride. A methylene
blue indicator strip is placed in the jar; methylene blue is blue in the
presence of oxygen and white when reduced. One of the
disadvantages of the Brewer jar is that the jar must be opened to
observe or use one plate. An inexpensive modification of the Brewer
jar has been developed using disposable plastic bags for one or two
culture plates (Figure. ). In this technique, the bag is coated with
antifogging chemicals, and a wet sodium bicarbonate tablet is added
to generate carbon dioxide. Iron (Fe°) activated with water removes O 2
as iron oxide (Fe2O3) is

produced. One plate can be observed without opening the bag.

3-Anaerobic incubators and glove boxes can also be used for incubation.
Air is evacuated from the chamber and can be replaced with a mixture
of carbon dioxide and nitrogen.

All methods of anaerobic culturing are only effective if the

specimen or culture of anaerobic organisms is collected and transferred
in a manner that minimizes exposure to oxygen. In this exercise, we will
try two methods of anaerobic culture and will perform the catalase test.

51
Materials
Petri plates containing nutrient agar (2)

Tubes containing thioglycollate both with indicator (4)

Brewer anaerobic jars (2 per lab section)

3% hydrogen peroxide (H2O2) (second period)

Cultures
Alcaligenes faecalis Enterococcus faecalis Escherichia coli Clostridium
sporogenes

Procedure
1-Don't shake the thioglycollaie. Why? What should you do to salvage it
if you do shake it?

2-Label four tubes of thiogl-ycollate broth and aseptically noculate one

with a loopful of Alcaligenes, one with Clost-ridium, one with
Enterococcus. and one with Escherichia.

3-Incubate at 35°C until the next lab period.

4-Record the appearance of growth in each tube.

5-With a marker, divide each nutrient agar plate into four sectors on the
bottom of the plate. Label one plate "Aerobic" and the other "Anaerobic."

6-Streak a single line of Alcaligenes, Clostridium, Ente-rococcus, and

Escherichia (Figure ).

7- Incubate the "Aerobic" plate, inverted, at 35°C until the next lab period.
Place the "Anaerobic" plate inverted, in the Brewer jar.

8-Your instructor will demonstrate how the jar is rendered anaerobic

once filled with plates Ten ml of water are added to a Gas-Pak(R)
generator and placed in the jar with a methylene blue indicator. What

52
causes the condensation that forms on the sides.of the jar?

9-Incubate the jar at 35°C until the next lab period.

10-After incubation, record the growth on each plate 11. Perform the
catalase test by adding a few drops of 3% H2O2 to the different colonies
on the nutrient agar. A positive catalase test produces a bubbling white
froth. A dissecting microscope (AppendixE) can be used if more
magnification is required to detect bubbling. The catalase test may also
be done by transferring bacteria to a slide and adding the H2O2 to it
Bacterial growth on blood agar must be tested this way. Why?

53
Question

-An organism is completely dependant on atmospheric O2 for growth. This organism

is a(n)

o A. Osmotolerant
o B. Acidophile
o C. Facultative anaerobe
o D. Aerotolerant anaerobe
o E. Obligate aerobe

54
 Growth: Osmotic Pressure and pH
Objectives
After completing this exercise you should be able to-
I-Define osmotic pressure and explain how it affects a cell.
2-Explain how microbial growth is related to osmotic, pressure and pH
3-Prepare and use a gradient plate
Background
The osmotic pressure of an environment influences microbial growth.
Osmotic pressure is the force with which a solvent moves from a
solution of lower solute concentration to a solution of higher solute
concentration across a semipermeable membrane (Figure ) The addition
of solutes, especially salts and sugars, and the resultant increase in
osmotic pressure, are used to preserve some foods. Can you name a
food that is pre- served with salt? -----------------------------

With sugar ----------------------

Bacteria are often able 10 live in a hypotonic (hypoosmotic)

environment in which the concentration of solutes outside the cell is
lower than inside the cell, However, when the concentration of solutes
outside the cell is higher than that inside the cell- a hypertonic
(hyperosmotic) envir-onment, a bacterial cell will undergo plasmolysis.
Plasmolysis occurs when water leaves the cell and the cytoplasmic
membrane draws inward, away from the cell wall. A few bacteria called
facultative halophiles, are able to tolerate salt concentrations up to
10%, and extreme halophiles require 15% to 20% salt.

The acidity or alkalinity (pH) of the environmentalso influences microbial

growth. Optimal bacterial growth usually occurs between pH 6.5 and pH

55
7.5. Only a few bacteria grow at an acidic pH below 4.0. Therefore,
organic acids are used to preserve foods such as fermented dairy
products (Exercise .).

Many bacteria produce acids that may inhibit their growth. Buffers are
added to culture media to neutralize these acids. The peptones in
complex media act as buffers. Phosphate salts are often used as buffers
inchemically defined media.

In this exercise, we will compare bacterial growth to fungal growth.

Fungi differ from bacteria morphologically and biochemically. The yeast
and mold used here are microbes belonging to the Kingdom Fungi.

Materials
Effect of Osmotic Pressure on Growth

(Your instructor will assign salt or sugar.)

Petri plates containing nutrient agar + 5%, 10%, 15%

NaCl (one of each) or Petri plates containing nutrient agar + 10%. 25%,
50% sucrose (one of each) or Petri plate containing nutrient agar

Gradient Plate

Sterile petri plate

tube containing melted nutrient agar

tube containing melted nutrient agar + 25% NaCl or 50% sucrose

salt or sugar enrichments

Effect of pH on Growth

Tubes containing nutrient broth adjusted to pH 2.5,5.0, 7.5, 9.5 (one of

each)

56
Cultures

Effect: of Osmotic Pressure on Growth

Escherichia coli
A yeast Saccharomyce
A mold (Pencillium)
Effect of pH on Growth
Staphylococcus aureus
Alcaligenes faecalis
Serratia marcescens
Escherichia coli
A mold (Penicillium)
A yeast (Saccbaromyces)
Procedure
Effect of Osmotic Pressure on Growth
1-Obtain a set of nutrient agar + salt or nutrient agar + sucrose plates.
Draw two crossed lines to form four quadrants on the bottom of one
plate. Each quadrant will be inoculated with one of the organisms and
should be labeled accordingly (Figure ). Repeat this procedure until all
plates are marked. One student group will mark the control plate nutrient
agar alone). What is the purpose of the control plate?

2-Using a sterile loop, inoculate S. aureus onto one quadrant on each

plate . Repeat this procedure to inoculate E. coli and the yeast.

3-Place a loopful of the mold suspension in the center of the remaining

quadrant on each plate

4-Invert the plates and incubate at 35°C for 24 to 48 hours. Record the
relative amounts of bacterial growth; the culture showing the "best"
growth is given (4 +) and others are evaluated relative to that culture.

57
5-Incubate the plate at room temperature for 2 to 4 days, and record
relative amounts of mold growth.

Gradient Plate
1-Pour a layer of nutrient agar into a sterile Petri plate and let it solidify,
with the plate resting on a small pencil or a loop handle (Figure ) so
that a wedge forms.

2-On the bottom of the plate, draw a line at the end corresponding to the
high side of the agar and label it "low." Draw four lines perpendicular to
the first line, and, if assigned sucrose, label the lines "0.5," "5," "10." and
"15"; if assigned salt, "0.5," "5," "15,"and "30" (Figure ).

3-Pour a layer of salt or sugar agar over the nutrient agar wedge. Let it
solidify. A solute gradient concentration has been prepared.

4-The following enrichments been prepared

a-Raisins were placed in tubes of nutrient of + 05.% sucrose, 5%

sucrose 10 % and 15% sucrose.

b-Hamburger was inoculated into tubes ofnutrient broth + 0 .5%

NaCl 5% NaCl 15 % NaCl and 30% NaCl

5. Using the enrichment with the same solute as agradient pleat streak a
loopful of each enrichment onto the( agar over the prelabeled lines from
the low to the high end

6. Incubate the plate inverted at 35°C for 48 °C and record the growth
patterns

Effect of pH on Growth
1. Inoculate a loopful of your organism into each of pH test broth

2. Incubate the tubes at 35°C for 24 to 48 hours, then record your

results and results to the agar tested by other students

58
59
Questions
A microbiology student noticed that a culture broth tube was very turbid at the surface but
clear throughout the rest of the tube. She can conclude that the ____________

o A. Organism are aerobes.

o B. Organism should be grown in an anaerobic chamber.
o C. Organism cannot produce superoxide dismutase and/or catalase.
o D. Organism cannot tolerate oxygen.
o E. Broth is sterile.

• 7.
Growth is defined as an increase in cellular constituents and may result in an increase in a
microorganism’s size, population number , or both

o A. TRUE
o B. FALSE

Extremophiles are microorganisms that grow under harsh or extreme environmental

conditions such as very high temperatures or low pHs.

o A. TRUE
o B. FALSE

• 17.
Generation time is the time required for a microbial population to double in number.

o A. TRUE
o B. FALSE

Which one of the following sequences exhibits increasing size

o A. Viruses to protozoa to bacteria

o B. Bacteria to viruses to fungi
o C. Fungi to protozoa to bacteria
o D. Viruses to bacteria to protozoa

60
 The gram stain technique is valuable in distinguishing

o A. Type of fungi
o B. The size and structure of viruses
o C. The nucleus of bacteria from other cellular organelles
o D. Types of bacteria

The solidifying agent commonly used in preparation of media is/are

A. agar

B. silica gel

C. both (a) and (b)

D. none of these
Answer: Option C
The concentration of agar to obtain semi solid media is
A. 1.5-20%

B. 0.5% or less

C. >10%

D. >10%but<20%
Answer: Option B

Mac-Conkey medium is an example of

A. transport medium

B. enrichment medium

C. differential medium

D. all of these
Answer: Option C

Agar is used for solidifying culture media because

A. it does not affect by the growth of bacteria

B. it does not add to the nutritive properties of the medium

C. the melting and solidifying points of agar solution are not the same

D. all of the above

61
Answer: Option D

The organism which grows best above 45°C are called

A. psychrophilic

B. mesosphilic

C. thermophilic

D. any of these
Answer: Option C

Lag phase is also known as

A. period of initial adjustment

B. transitional period

C. generation time

D. none of these
Answer: Option A

The growth is normally expressed as __________ in turbidimetric measurement

A. cells per ml

B. cfu/ml

C. optical density

D. mg N2 /ml
Answer: Option C

A culture broth tube was very turbid at the surface but clear throughout the rest of the tube
indicating that the
A. organism are aerobes

B. organism should be grown in an anaerobic chamber

C. organism cannot produce superoxide dismutase and/or catalase

D. organism cannot tolerate oxygen

Answer: Option A
62
33. The term facultative anaerobe refers to an organism that
A. doesn't use oxygen but tolerates it

B. is killed by oxygen

C. uses oxygen when present or grows without oxygen when absent

D. requires less oxygen than is present in air

Answer: Option C
The microorganisms that grow best in a low-oxygen environment is called a (n)
A. aerobe

B. anaerobe

C. facultative

D. microaerophile
Answer: Option D

Which of the following is the suitable temperature range for mesophiles?

A. 20-30°C

B. 25-40°C

C. >40°C

D. None of these
Answer: Option B

In the exponential phase, the cells and cell mass

A. first increases then decreases

B. decreases

C. are constant

D. double at a constant rate

Answer: Option D

For most bacteria, the optimum pH for growth lies between

A. 6.5-7.5

B. 3.5-4.5

C. 4.5-5.5

63
 D. 5.5-6.5
Answer: Option A

The cell mass can be measured optically by determining the amount of light scattered by a
suspension of cells. The measurements are usually at a wavelength of
A. 300-400nm

B. 400-500nm

C. 500-600nm

D. 600-700nm
Answer: Option D

64
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Allen, James, J. (1976). Light Microscopic Techniques in Biology and Medicine (Netherlands:
Martinus
Nijhof).

Pluta, M. (1993). Advanced Light Microscopy, vol. 3, Measuring Techniques (Amsterdam:

Elsevier).

rd
Henrici, A. T. The biology of bacteria 3 (D. C.
Heath & Co., Boston, 1948).

Caldwell, D. R. (2000). Microbial Physiology and Metabolism, 2nd edn. Belm, CA: Star
Publishing

Dawes, I. W. & Sutherland, I. W. (1992). Microbial Physiology, 2nd edn. Basic Microbiology
Series, 4. Oxford: Blackwell.

Gottschalk, G. (1986). Bacterial Metabolism, 2nd edn. New York: Springer-Verlag.

Ingraham, J. L., Maaloe, O. & Neidhardt, F. C. (1983). Growth of the Bacterial Cell. Sunderland,
MA: Sinauer Associates

Mandelstam, J., McQuillin, K. & Dawes, I. (1982). Biochemistry of Bacterial Growth, 3rd
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Moat, A. G., Foster, J. W. & Spector, M. P. (2002). Microbial Physiology, 4th edn. New York:
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Laboratory Manual in Microbiology. The Sri Lanka College of Microbiologists. 2nd Edition
2011. 1.

Performance standards for antimicrobial susceptibility testing; Twenty second informational

supplement M100-S22, F. R. Cockerill, M. A. Wikler, J. Alder et al, Vol. 32 No.3, Clinical
Laboratory Standards Institute, January 2012

Whitford, L. A., and G. L. Schumacher. 1973. A Manual of Fresh-water Algae. Sparks Press,
Raliegh.

Wołowski, K., and F. Hindák, 2008. Atlas of Euglenophytes. VEDA, Bratislava Round, F. E., R.
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