1. 1
Chapter 3 : Techniques in
microbiology

2. BIO 300
BIOLOGICAL TECHNIQUES AND SKILLS
SARINI BINTI AHMAD WAKID
FACULTY OF APPLIED SCIENCE
CHAPTER 3
TECHNIQUES IN MICROBIOLOGY
Chapter 3 : Techniques in
microbiology
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3. What is Microbiology?
The science and study of microorganims
What is Microorganisms?
Microorganisms are minute living things that
individually are usually too small to be seen with
unaided eye. There are four major kinds of
microbes: bacteria, fungi, protists and viruses.
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4. History and scope of microbiology
The Importance of Microorganisms
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medical and most populous group of organisms and are
found everywhere on the planet
play a major role in recycling essential elements
source of nutrients and some carry out photosynthesis
benefit society by their production of food, beverages,
antibiotics and vitamins
causative agents of some important diseases
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5. Microorganisms:
- Microorganisms are everywhere; almost every natural surface is colonized by
microbes, from body to ocean. Some microorganisms can live hot springs, and others in
frozen sea ice.
- Most microorganisms are harmless to humans; You swallow millions of microbes
every day with no ill effects. In fact, we are dependent on microbes to help us digest
our food.
- Microbes also keep the biosphere running by carrying out essential functions such as
decomposition of dead animals and plants. They make possible the cycles of carbon,
oxygen, nitrogen and sulfur that take place in terrestrial and aquatic systems.
- Microorganisms have also harmed humans and disrupted society over the millennia.
-They sometimes cause diseases in man, animals and plants.
-They are involved in food spoilage.
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6. - Organisms divided into 5 Kingdoms:
• Monera – all procaryotes
• Protista – unicellular or colonial eucaryotic cells lacking true tissues; includes
algae, protozoa & simpler fungi
• Fungi – eucaryoutic; includes molds, yeasts and mushrooms
• Plantae – multicellular
•Animalia - multicellular
• Scope of Microbiology:
- Microbiology has an impact on medicine, agriculture, food science, ecology, genetics,
biochemistry, immunology, and many other fields.
- Many microbiologists are primarily interested in the biology of microorganisms,
while others focus on specific groups;
- Virologists - viruses
- Bacteriologists - bacteria
- Phycologists – algae
- Mycologist -fungi
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7. - Medical Microbiology: deals with diseases of humans and animals; identify and plan
measures to eliminate agents causing infectious diseases.
- Immunology: study of the immune system that protects the body from pathogens.
- Agricultural Microbiology: impact of microorganisms on agriculture; combat plant
diseases that attack important food crops.
- Food and Dairy Microbiology: prevent microbial spoilage of food & transmission of
food-borne diseases (e.g. salmonellosis); use microorganisms to make food such as
cheeses, yogurts, pickles, beer, etc.
- Industrial Microbiology: using microorganisms to make products such as
antibiotics, vaccines, steroids, alcohols & other solvents, vitamins, amino acids,
enzymes, etc.
- Genetic Engineering: Engineered microorganisms used to make hormones,
antibiotics, vaccines and other products.
- Since viruses are acellular and possess both living and nonliving characteristics, they
are considered neither prokaryotic nor eukaryotic. They will be discussed in separate
section of the course.
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8. Classification of microbes
Bacteria
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9. BACTERIA

ARCHAEBACTERIA


Lack peptidogycan in cell
walls
Live in extreme
environments

EUBACTERIA
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Includes most bacteria
Most have one of three
shapes
May be divided into up to
12 phyla
Classification is
controversial
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10. TYPES OF ARCHAEBACTERIA
Methanogens
living in sewage
Thermoacidophilies
Living in hot springs
Extreme halophile
living in the Great Salt Lake
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11. Archaebacteria

Live in extreme
locations:
Oxygen-free
environments
 Concentrated
salt-water
 Hot, acidic
water

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12. Eubacteria - Heterotrophs
 Found
everywhere
 Parasites: live off of other
organisms
 Saprobes: live off of dead
organisms or waste (recyclers)
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13. Eubacteria: Photosynthetic Autotrophs
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Photosynthetic: make their own food from
light
Cyanobacteria: blue-green, yellow, or red
ponds, streams, moist areas
Eubacteria: Chemosynthetic Autotrophs
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Get energy by breaking down inorganic
substances like sulfur and nitrogen
Make nitrogen in the air usable for plants
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14. Structure of Bacteria
 Two
parts to Bacteria Structure:
Arrangement
 Shape

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15. Arrangement
 Paired:
diplo
 Grape-like clusters: staphylo
 Chains: strepto
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16. Shape
 Rod:
bacillus
 Spheres: coccus
 Spirals: spirillum
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17. Examples
Streptococcus: chains of spheres
 Staphylospirillum: Grapelike
clusters of spirals
 Streptobacillus: Chains of rods

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18. BASIC SHAPES OF EUBACTERIA
ROD-SHAPED
SPHERICAL
SPIRILLA
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19. Most Species of Eubacteria may be
Grouped Based on Staining

Gram-Negative
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Lack thicker layer of
peptidoglycan
Stain pink
Endotoxins
Gram-Positive

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Thicker layer of
peptidogycan
Stain purple
Exotoxins (released
when bacteria die)
Gram-positive
Gram- negative
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20. Classification of microbes
FUNGI
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21. WHAT ARE FUNGI?

Fungi are not classed as animals or plants,
they have a Kingdom of their own to which they
belong.

They range from being just a single cell, like
the yeasts, to others that cover hundreds of
acres of land.

Most fungi are said to be filamentous. This is
because the main body of the fungus is made
up of thin, thread-like filaments that are called
hyphae, which form the mycelium.
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22. KINGDOM FUNGI
To date, 100,000 species of
fungi have been discovered.

People that study fungi are called
Mycologists.
It is thought that there are over
one million species still to be
found.

Fungi are not able to produce their
own food as plants do.

Fungi are said to be
SAPROTROPHS, because they live
on dead organic matter such as
leaves and wood.

To obtain nutrients fungi secrete
special digestive enzymes which
degrade organic material outside the
mycelium. The degraded
compounds can then be ingested.
The fungi that most people are
familiar with are those that form
fruit bodies or mushrooms.
Fungi can live in many habitats
including the arctic, tropical
rainforest, fresh and salt water.
However, most fungi live in soil.
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23. Traits of Fungi

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They are either:
 Saprobes – feed on material from previously living things (shoes,
dead trees, dead animals etc.) or
 Parasites – which eat or derive there energy from living things.
To reproduce, they
 send out spores instead of seeds.
 Carry pieces of broken hyphae to new places
 Form Buds in which a small part of the parent grows into a new
organism.
.
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24. Traits of Fungi

Most are multicellular

Some like yeasts are unicellular
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25. Examples of Fungi

Bread Mold
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26. What are True FUNGI?
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Eukaryotic organisms
Heterotrophic, lacking chlorophyll
Obtain nutrients via enzyme secretion and
absorption of resulting byproducts
Cells walls containing chitin and beta glucans
Glycogen as primary food storage
Can reproduce both sexually and asexually
Heterotrophic – as such can consume almost any
carbonaceous substrate including jet fuel and wall
paint
Biggest role is in the recycling of dead plant material
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27. From The Fungi Name Trail by Liz Holden & Kath Hamper
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28. Chapter 3 : Techniques in microbiology
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29. Chapter 3 : Techniques in microbiology
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30. Fungal Ecology
Saprobe
decomposer of all terrestrial organic
matter (and some aquatic matter)
Pathogen
purveyor of plant and animal disease
Mycorrhizae
symbiosis of plant and fungus (fungi)
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31. Mycorrhizae

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The term mycorrhiza, which
literally means fungus-root
first applied to fungus-tree
associations described in 1885
95% of all plant species
Symbiotic associations that
form between the roots of most
plant species and fungi
characterized by bi-directional
movement of nutrients where
carbon flows to the fungus and
inorganic nutrients move to the
plant
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32. Helpful Fungi
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Food – mushrooms
Used to make cheese – Blue Cheese
Used to make wine, beer, and whiskey (Yeast)
Used to make bread rise
Used to make soy sauce from soy beans
Used to break down materials and recycle wastes
and dead organisms
Used to make certain drugs (ex. Penicillin)
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33. Harmful Fungus
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Cause food spoilage
Cause plant disease such as rusts, Dutch
Elm Disease, and mildew
Cause Human diseases such as Ring Worm,
Athlete’s Foot, Thrush, lung Infections, and
Yeast Infections
Destroy leather, fabrics, plastics, etc.
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34. Food Spoilage
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35. Ringworm
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36. Athlete’s Foot – Tinea pedis
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37. Thrush
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38. Fungal Lung Infection
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39. Yeast Infections
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40. Fungus Destroying Leather
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41. Classification of microbes
ALGAE
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42. Characteristics
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Range in size from microscopic to single
celled organisms to large seaweed
Autotrophic
Form the reproductive structures –
gametangia or gamete chambers
Aquatic and have flagella at some point in life
Often contain pyrenoids, organelles that
synthesis and store starch
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43. IDENTIFY THE TYPE OF ALGAE
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44. ALGAE
MICROALGAE
MACROALGAE
Unicellular
-body is only comprised of one cell
Multicellular
-differentiated structures within cells
to perform photosynthesis, flotation,
anchorage and others.
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45. Classification of algae
Algae are classified into seven major groups:
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Chrysophyta (golden brown algae)
Cyanobacteria (blue green algae)
Pyrrophyta (dinoflagellates)
Euglenophyta (Euglenoid)
Rhodophyta (red algae)
Chlorophyta (green algae)
Phaeophyta (brown algae)
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46. BACILLARIOPHYCEAE
Examples: Chaetoceros sp., Coscinodiscus sp., Asterionella
sp., Cymbella sp., Frustulia sp.
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47. CHRYSOPHYCEAE
Examples: Dinobryon sp., Synura sp.,
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48. CYANOBACTERIA
Examples: Anabaena sp., Oscillatoria sp., Nostoc sp.,
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49. PYRROPHYTA
Examples:Peridinium sp., Ceratium sp.
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50. Phylum Euglenophyta


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1000 species of
Euglenoids
Have both plantlike and
animal-like
characteristics
Fresh water
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51. EUGLENOPYHTA
Examples: Euglena sp., Phacus sp., Lepocinclis sp.,
Strombomonas sp.
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52. RHODOPHYTA
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Some are single-celled, others are
macroscopic and multicellular.
Mostly marine algae
The larger species typically grow attached to
a hard substrate or occur as epiphytes on
other algae.
Contain chlorophyll a and d, but appear red
due to accessory pigments,phycocyanin and
phycoerythrin.
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53. RHODOPYHTA
Examples: Porphyra sp., Batrachospermum sp
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54. CHLOROPHYTA
Examples: Cosmarium sp., Closterium sp., Spirogyra sp.,
Caulerpa racemosa.
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55. “Kelp Forests”
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56. USES O ALGAE
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Agar
Alginates
Energy source - Biofuel
Fertilizer
Nutrition – Health food
Pollution control – treatment sewage
Pigments – chemical dyes, coloring agents.
Stabilizing subtances
Cosmetics
Animal Feed
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57. Classification of microbes
VIRUS
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58. INTRODUCTION TO VIRUSES
 Virus means "veleno". Viruses are basically a
way a form of genetic information insures its
continued survival. They are entities which
reproduces their DNA/RNA within living cells
utilizing mechanisms of cells for this.
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59. VIRUS COMPOSITION

Viruses are unique from all other life forms in that they can contain
ONLY ONE FORM OF NUCLEIC ACID. Some viruses use RNA as their
genetic material and other use DNA, but NEVER do they contain both.
Further, this nucleic acid polymer may either exist as DOUBLE
STRANDED (DS) DNA or RNA or as SINGLE STRANDED (SS) DNA or
RNA. Each of these characteristics is a constant for a particular virus
and is part of it description. The nucleic acid polymer may contain as
few as 4 to 7 genes for very small viruses to 150 to 200 genes for very
large viruses. In some viruses the nucleic acid exists in more that one
molecule. Some viruses contain a few enzymes and some contain
none, but no viruses contain the large numbers of enzymes found even
in the smallest bacteria.
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60. 
All virus are covered with a PROTEIN COAT. This protein coat is mainly
composed of a FEW TYPES of proteins of which there are many copies
per virus; something like the individual threads in a shirt. These identical
protein subunits are called CAPSOMERES and they are made so that
they spontaneously come together (ASSEMBLE) in a
PREDETERMINED way to produce the virus coat which is called the
CAPSID.

If a virus has ONLY a protein capsid covering it, it is termed a NAKED
CAPSID VIRUS or a NAKED VIRUS. However, some viruses pick up a
lipid membrane from the host cell when it is released, that surrounds the
capsid. The lipid membrane is called an ENVELOPE and such viruses
are termed ENVELOPED VIRUSES.
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61. Virus Structure

Size


Basic shape

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Rod-like
“Spherical”
Protective Shell - Capsid

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
17 nm – 3000 nm diameter
Made of many identical protein subunits
Symmetrically organized
50% of weight
Enveloped or non-enveloped
Genomic material


DNA or RNA
Single- or double-stranded
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62. Virus Structure

Virus capsids function in:


Packaging and protecting nucleic acid
Host cell recognition


Protein on coat or envelope “feels” or “recognizes” host
cell receptors
Genomic material delivery


Enveloped: cell fusion event
Non-enveloped: more complex strategies & specialized
structures
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63. Chapter 3 : Techniques in microbiology
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64. Culture Media
Culture is the term given to microorganisms that are cultivated in
the lab for the purpose of studying them.
Medium is the term given to the combination of ingredients that
will support the growth and cultivation of microorganisms by
providing all the essential nutrients required for the growth
(that is, multiplication) in order to cultivate these
microorganisms in large numbers to study them.
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65. Laboratory culture: pure culture
- Contaminants = other microorganisms present in the sample
- history of the pure culture:
- Koch employed gelatin as solidifying agent
- Walter Hesse adopted agar
- Petri (1887) invented Petri-dish
- culture medium:
- rich/selective
Confluent mixture
- growth inhibitors
1
Isolated colony
- liquid/solid
- temperature
-Nutrients:
- carbon, nitrogen, elements ...
-Aseptic technique:
- sterilization of medium and equipment
4
- proper handling
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3
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66. Among the different kinds of microorganisms the two groups that
can be grown in cultures are bacteria and fungi.
Algae and protozoa require many different nutrients in minute
quantities that are difficult to anticipate and prepare in the lab.
These organisms have different nutritional requirements and
thus various kinds of culture media have been developed.
Primary ingredients required by all living organisms include:
a carbon source, water, minerals, and a nitrogen source.
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67. Living cells need nutrients required for their structure (biosynthesis)
as well as nutrients to provide them with energy to perform all
of their various life processes.
Nutrients are acquired from the environment in which they live in
their natural habitat.
Most of these nutrients are then processed within the cell through a
variety of metabolic pathways to be incorporated in different
ways.
The process of building complex structures from simple building
blocks is called anabolism.
The process of breaking up complex materials to harvest the energy
in them is called catabolism.
The ability to use particular compounds is dependent upon the
genetic makeup (DNA) that the cells have.
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68. Since there are different kinds of organisms that can be grown in
culture media with varying needs, culture media have also
been formulated with different ingredients.
Culture media may be found in one of three states:
liquid (called broth)
semi-solid
solid.
Media are solidified by the addition of solidifying agents such as
agar (inert compound).
Varying the concentration of agar will yield varying degrees
of solidification.
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69. Culture media may be classified as:
Synthetic media (Defined)
Complex (Non-synthetic) media
Synthetic media contain only ingredients for which a complete
chemical formula is known.
Complex media contain at least one ingredient for which a chemical
formula is not known (such as milk, egg, malt, animal tissues)
Culture media can also be classified based on the function they
perform in determining various characteristics of organism
that are able to grow on/in them
e.g. Differential, Selective media.
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70. Microbial growth media
- chemically defined: highly purified inorganic and organic compounds in dest. H2O
- complex (undefined): digests of casein, beef, soybeans, yeast, ...
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71. Microbial growth media
Media
*Complex
Purpose
Grow most heterotrophic organisms
*Defined
Grow specific heterotrophs and are often mandatory for
chemoautotrophs, photoautotrophs and for microbiological
assays
*Selective
Suppress unwanted microbes, or encourage desired microbes
*Differential
Distinguish colonies of specific microbes from others
*Enrichment
Similar to selective media but designed to increase the numbers
of desired microorganisms to a detectable level without stimulating
stimulating the rest of the bacterial population
*Reducing
Growth of obligate anaerobes
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72. Microbial nutrition
Nutrients = chemical „tools“ a cell needs to grow/replicate
Macronutrients = chemicals needed in large amounts
Micronutrients = chemicals needed in small/trace amounts
Autotrophy = CO2 can be sole C-source
% of dry
weight
50%
12%
(sometimes non-essential)
(sometimes non-essential)
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73. Media Provides Nutrients for Bacteria

Nutrient broth: liquid media (trypticase soy broth,
TSB)

Nutrient agar: solid media (trypticase soy agar,
TSA)



Agar slants (tubes)
Agar plates
Agar: polysaccharide isolated from red algae

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Solid at room temp (25oC)
Liquid at 100oC
Provides framework to hold moisture & nutrients
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74. Microbial nutrition: Growth factors
- organic compounds required
by some bacteria
- vitamins, amino acids, purines,
pyrimidines
- Streptoccus, Lactobacillus,
Leuconostoc (lactic acid
bacterium):
complex vitamin
requirements
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75. Chapter 3 : Techniques in microbiology
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76. The ingredients in a medium will affect the chemical nature of the
medium.
This is important because organisms vary in their requirement for
different environments.
One such property is:
pH (which is a measure of the amount of hydrogen ions in
a particular medium).
This has to be monitored during the preparation of media since this
will influence the kind of organisms that are able to grow in the
medium.
The pH of the medium will thus determine which organisms are
able to grow on the medium.
For example, fungi prefer acidic media for their growth while bacteria
grow on neutral pH media.
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77. The primary function of culture media is to be able to grow
particular organisms on/in them.
It is important that these media are devoid of any other living
organisms.
This is possible through the process of sterilization
(a process by which all living organisms and their spore forms
are killed and the medium is made sterile)
Culture media are most commonly sterilized through the process of
autoclaving (using high temperatures that will kill all living
organisms under increased pressure for specified periods of time –
in an appliance called the autoclave)
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78. Chapter 3 : Techniques in microbiology
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79. Aseptic Techniques
…protective clothing
…hand washing
…bench cleaning
…loop flaming
…pipettors
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80. The 5 I’s of Culturing Microbes
1.
2.
3.
4.
5.
Inoculation: introduction of sample into a
container of media
Incubation: under conditions that allow growth
Isolation: separating 1 species from another
Inspection
Identification
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81. Inoculation
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82. Incubation & Isolation
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83. Pure vs Mixed Cultures

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Eschericia coli (white)
Micrococcus luteus (yellow)
Serratia marcescens (red)
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84. Isolation Technique
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85. Chapter 3 : Techniques in microbiology
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86. Inspection & Identification
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87. Bacteria
1) Bacillus = Rod shaped. (pl. bacilli)
(diplobacilli, streptobacilli)
2) Coccus = Round shaped. (pl. cocci)
(diplococci, streptococci, staphylococci)
3)Spiral = Spiral shaped
(spirilla, vibrio, spirochete)
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88. Bacteria may appear as single cells or
in groups:

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These terms describe typical bacteria
groupings:
1) diplo = paired cells
2) strepto = long chains
3) staphylo = grape-like clusters
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89. Bacterial growth
Growth rate = ∆cell
number/time
or ∆cell mass/time
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1 generation
Growth = increase in # of cells
(by binary fission)
generation time: 10 min - days
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90. Bacterial growth: exponential growth
Generation time = 30 min
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91. Bacterial growth: exponential growth
Semilogarythmic plot
Straight
line
indicates
logarithmic
growth
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92. Bacterial growth: calculate the generation time
t
g= n
t = time of exponential growth (in min, h)
g = generation time (in min, h)
n = number of generations
The generation time is the time needs the
culture population to double
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93. Bacterial growth: calculate the generation time
t
g= n
t = time of exponential growth (in min, h)
g = generation time (in min, h)
n = number of generations
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94. Bacterial growth: calculate the generation time
t = time of exponential growth (in min, h)
g = generation time (in min, h)
n = number of generations
t
g= n
Nt = N0 x 2
n
Nt = number of cells at a certain time point
N0 = initial number of cells
n = number of generations
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95. Bacterial growth: calculate the generation time
t = time of exponential growth (in min, h)
g = generation time (in min, h)
n = number of generations
t
g= n
Nt = N0 x 2
Nt = number of cells at a certain time point
N0 = initial number of cells
n = number of generations
n
logNt = logN0 + n x log2
logNt - logN0= n x log2
n=
logNt - logN0
log2
n = 3.3 x (logNt - logN0)
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96. Bacterial growth: batch culture
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97. Batch culture: Lag phase
no Lag phase:
Inocculum from exponential phase grown in the same media
Lag phase:
Inocculum from stationary culture (depletion of essential constituents)
After transfer into poorer culture media (enzymes for biosynthesis)
Cells of inocculum damaged (time for repair)
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98. Batch culture: exponential phase
Exponential phase = log-phase
Maximum growth rates
„midexponential“: bacteria often used for functional studies
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99. Batch culture: stationary phase
Bacterial growth is limited:
- essential nutrient used up
- build up of toxic metabolic products in media
Stationary phase:
- no net increase in cell number
- „cryptic growth“
- energy metabolism, some biosynthesis continues
- specific expression of „survival“ genes
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100. Batch culture: death phase
Bacterial cell death:
- sometimes associated with cell lysis
- 2 Theories:
- „programmed“: induction of viable but non-culturable
- gradual deterioration:
- oxidative stress: oxidation of essential molecules
- accumulation of damage
- finaly less cells viable
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101. Measurement of microbial growth
A. Weight of cell mass
B. number of cells:
- Total cell count
- Viable count
- Dilutions
- turbidimetric
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102. total cell count
A. Sample dried on slide
B. Counting chamber:
Limitations:
- dead/live not distinguished
- small cells difficult to see
- precision low
- phase contrast microscope
- not useful for < 106/ml
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103. viable cell count
synonymous: plate count, colony count
1 viable cell  1 colony
cfu = colony forming unit
Advantage: high sensitivity; selective media
Optimal: 30 – 300 colonies per plate ( plate appropriate dilutions)
spread plate method:
pour plate method:
Bacteria must withstand 45°C briefly
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104. dilutions
Example:
3 h culture of E. coli in L-broth
How do I determine the actual number?
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105. Turbidimetric measurements
Relationship between OD and cfu/ml must be established experimentally
Exponential culture of E. coli in L-broth: 1 OD = ca. 2-3 x 109 cfu/ml
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106. Continuous culture: the chemostat
steady state = cell number, nutrient status remain constant
Control:
1. Concentration of a limiting nutrient
2. Dilution rate
3. Temperature
 Independent control of:
- Cell density
- Growth rate
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107. Factors affecting microbial growth
•
•
•
•
•
Nutrients
Temperature
pH
Oxygen
Water availability
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108. NEXT CLASS:
Chapter 4
TECHNIQUES IN BIOCHEMICAL
ANALYSIS
THANK YOU
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