Lecture Note of General Microbiology

1. Basic methods of microbiology
Chapter Two
BY: Abu Feyisa (Abu Feyisa (M.Sc. Applied Microbiology,
Ph.D. Candidate Industrial Biotechnology)

2. 2.1. Theories and practices of sterilization
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The Sterilization Methods
• Control of microorganisms is essential in the
home, industry and
medical fields
prevent and treat disease
What is the purpose of sterilization?
 to inhibit the spoilage of foods and other industrial products
What are the common method of sterilization?
 chemical agents and
 physical agents that affect microbial structures and functions

3. How???
by producing a microbicidal or microbistatic
 Microbicidal: agents kill microorganisms
 Microbistatic: agents inhibit microbial growth and multiplication
Bactericidal - kills bacteria
Bacteristatic - inhibits bacterial growth
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Terms used
1. Sterilization vs. Disinfection
• Sterilization
 destroying all forms of life
• Disinfection
 destroying pathogens or unwanted organisms
2. Disinfectant vs. Antiseptic
• Disinfectant
 antimicrobial agent used on inanimate objects
• Antiseptic
 antimicrobial agent used on living tissue

4. 4
• Meaning “against life,” to describe the group of drugs that had action
against microorganisms.
• It can be inhibited the growth or kill microorganism
Antimicrobial Activity

5. 5
Targets of antimicrobial agents
• Antimicrobial agents have been classified based on their mechanism
of action, chemical structure, or spectrum (broad spectrum or narrow
spectrum) of activity.
1. Inhibit cell wall synthesis
2. Disruption of cell membrane structure and function
3. Inhibit enzymes & proteins synthesis
4. Inhibit DNA & RNA synthesis
5. Blocks metabolic pathways
Factors that effect antimicrobial activity
• Temperature
• Time/duration of exposure
• Concentration of antimicrobial agent
• Type of microbe
• Presence of organic matter

6. 1. Physical Methods of Microbial Control
1. Heat: moist heat, dry heat
• kills by denaturing and coagulate enzymes and proteins
A. Thermal Death Point (TDP)
• lowest temperature at which all microorganism in a liquid culture
are killed in 10 minutes
B. Thermal Death Time (TDT)
• minimum length of time in which all microorganisms in a liquid
culture are killed at a given temperature
I. Moist Heat
A. Boiling Water
• kills vegetative bacterial cells, Fungi and many viruses
• not effective for endospores and some viruses
• Hepatitis (20 min)
• Some spores may survive boiling water for up to 20 hrs
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7. B. Autoclave (Steam under pressure)
• is a preferred method of sterilization
• Water boils at 100 0C, will not be heated to >100 0C.
• But Increasing the pressure raises the Temp.
• So autoclaving requires 121 0C for 15 min at high pressure
II. Dry Heat
1. Direct Flaming
• Inoculating Loop and Needle 100% effective
2. Incineration
• disposable wastes (paper cups, bags, dressings)
3. Hot Air Sterilization
• Oven ( 170 0C for 2 hours)
• used on substances that would be damaged by moist heat sterilization
• gauzes, dressings or powders
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9. II. Filtration
• Removes microorganisms from solutions that might be
damaged by heat
• enzymes
• vaccines
• antibiotics
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10. III. Radiation
• 1. Ionizing Radiation
• Gamma (γ ) rays from the isotope cobalt 60 (60Co) & x-rays
• penetrates most substances
• Ionizing radiations have a shorter wavelength and much higher
energy, giving them greater penetrating powers.
• Produce highly reactive free radicals, which disrupt the structure
of DNA and proteins.
• Used on substances that could be damaged by heat
• plastic Petri dishes
• plastic syringes
• catheters
• surgical gloves
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11. 2. Non-Ionizing Radiation
• UV Light
• Wavelengths around 260nm are used because these are absorbed by
the purine and pyrimidine components of nucleic acids(thymine
Dimers), as well as certain aromatic amino acids in proteins.
• The absorbed energy causes a rupture of the chemical bonds, so that
normal cellular function is impaired.
• does not penetrate plastic, glass or proteinaceous matter
• Used to reduce microbial populations in
• hospital rooms
• nurseries
• operating rooms
• Laminar floe hoods
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12. 2. Chemicals Methods of Microbial Control
• Ethylene Oxide = alkylating agent
• chemically adds itself on to proteins, nucleic acids, etc. and affects
the structure of both proteins and nucleic acids
• formaldehyde, hydrogen peroxide
• Widely used (in special cabinets) to sterilize heat-sensitive items, e.g.
syringes, disposable plastics, rubber items, surgical supplies, etc.
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13. 13
IV. Pasteurization
• Disinfection - not sterilization (removes unwanted organisms)
• Mycobacterium tuberculosis
• 63 0C for 30 minutes
• 72 0C for 15 seconds
• Thermoduric microbes cannot be killed.
• able to survive high temps.

14. • heavy metals
• (Mercury, Silver, Arsenic)- cause protein denaturation
• halogens (Chlorine, Iodine, Hypochlorite)-
• oxidizing agents.
• Not usually used as antiseptics, but good for swimming pools,
hot tubs, water supplies.
• Household bleach = 5% soln of hypochlorite- good for all-
purpose disinfectant.
• phenols & cresols- dissolve membranes, denature proteins
• alcohols- denature proteins, dissolve membranes.
• detergents- dissolve membranes
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15. 2.2. Microbiological Culture Media and its preparation
2.2.1. Culture media
 Culture: Microbes that grow and multiply in or on a culture medium.
 Media: Nutrient material prepared for microbial growth in laboratory.
• Are solutions containing all of the chemicals and elements necessary for microbial
growth, which are referred to as nutrients or nutritional requirements.
• Are mixtures of nutrients for growth of mos. outside their natural habitat.
• They supply the organism with sources of energy and elements allowing its optimum
growth.
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16. Media Preparation

17. • Culture media must include all the following
requirements for growth
- Water
- Nitrogen-containing compounds
- Energy sources
- Accessory growth factors
- Solidifying agents
- pH Indicators
- Reducing agents
- Selective agents
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18. A- Water:
 to dissolve all the material components.
 distilled water is preferred in use because the tap water may contain
calcium and magnesium salts which can react with other ingredients
and can inhibit the growth of microorganisms.
B- Nitrogen-containing compounds:
• in most cases peptone is used as nitrogen source
• 3 examples of brand names of peptones are
* Tryptone (or Trypticase; a pancreatic digest of casein),
* Phytone (or Soytone; digest of soybean meal)
* Peptone (a digest of beef muscle).
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19. • Peptones has the following functions in the medium:
1. is the constituent of amino acids, nucleic acids nucleotides,
and coenzymes,
2. is a buffering material; because it is an amphoteric
compound it can maintain the medium pH,
3. in conjunction with extracts, peptones are used frequently
for the cultivation of fastidious organisms.
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20. C- Energy sources:
• Carbohydrates like glucose (dextrose), peptides, amino acids and
proteins are used to provide energy for metabolic activity of microbe.
D- Accessory growth factors
• extracts are used as a source of the growth factors.
• extract are eucaryotic tissues (yeast, beef muscle, liver, brain, heart,
etc.) extracted by boiling and then concentrated to a paste or dried to
a powder.
• infusion (liver, brain, heart infusion) refers to the aqueous extracts
of these materials used for same purposes without being dried or
otherwise concentrated.
• Beef extract source of amino acids, vitamins and coenzymes, needed
as growth factors by fastidious organisms
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21. • Yeast extract rich in carbohydrates and vitamins.
• Malt extract prepared from barley grains.
• It is rich in maltose, glucose and dextrin.
• Whole blood, plasma, serum or other body fluids are added to
culture media for the isolation and cultivation of many pathogens.
• Body fluids contribute many growth factors and/or substances
which detoxify certain inhibitors.
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22. E- Solidifying agents:
* Are agents used only in the solid reversible to liquid media.
* Gelatin and agar are the most frequently used agents.
Gelatin:
• a protein added to the medium in a concentration of 10-12% of the
medium.
• melts at 25-300C and solidifies at below 250C.
• if heated to ~1000C or above; it will melt and cannot solidify again.
• is digestable by most proteolytic microbes, therefore used to test for
their proteolytic activity.
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23. Agar:
• a carbohydrate which is added in a concentration of 1.5 – 2% w/v
(1.5-2.0g/100 ml) to the culture medium.
• It melts at 84°C and solidifies at 38°C-45°C.
• is the predominant solidifying agent because:
1. most bacterial strains can solidify gelatin, but not agar
2. gelatin can be molten at the incubation temperature, agar does
not liquefy again until it is heated
3. agar is not digestible by the vast majority of microorganisms
but gelatin does.
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24. Other ingredients for special purposes in the media include:
• pH Indicators:
* acid-base indicator
* added to differential media to detect changes
in hydrogen ion concentration during the
growth of an organism
Example: commonly used indicator
• Methyl Red: pH range 4.2 – 6.3 Red Yellow
• Bromcresol purple: pH range 5.2 – 6.8, Yellow Purple
• bromothymol blue: PH range 6.0 – 7.6, Yellow Blue
• phenol red: 6.8 – 8.4 Yellow Red
• Phenolphthalein 8.3 – 10.0 Colorless Red
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25. Reducing agents:
* certain chemicals may stimulate growth by reducing the
environment .
* Cysteine & thioglycollate are reducing agents often used for the
cultivation of anaerobes.
• found in the thioglycollate Medium utilized to determine
oxygen relationship.
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26. Selective agents
* Antimicrobial agents such as crystal violet, bile salts, brilliant
green, potassium tellurite, sodium azide and antibiotics can be
employed in selective media.
• These agents suppress or inhibit the growth of certain groups of
microbes while allowing growth of desired organisms.
* These agents are usually bacteriostatic.
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27. 2.2.2. Types of Culture Media
I. Based on their consistency
a) Solid medium
b) Liquid medium
c) Semi solid medium
II. Based on the constituents/
ingredients
a) Simple medium
b) Complex medium
c) Synthetic or defined medium
d) Special media
d. Special media
• Enriched media
• Enrichment media
• Selective media
• Indicator media
• Differential media
• Sugar media
• Transport media
IV. Based on Oxygen requirement
- Aerobic media
- Anaerobic media

28. 28
Solid Media
• Nutrient material that contains a solidifying agent.
• common solidifying agent are agar and gelatin.
Defined Media
• Nutrient material whose exact chemical composition is known.
Complex Media
• Nutrient material whose exact chemical composition is not
known.
Example:
1. Nutrient agar
2. Nutrient broth

29. Enriched Media
• Contain nutrients (growth factors) required to support the growth of
a wide variety of organisms, including some of the more fastidious
ones.
• These factors are usually body fluids .
Example:
1. Blood Agar
2. Chocolate Agar (Diplococcus pneumoniae)
3. Loeffler’s Serum (Corynebacteria , shows proteolytic activity)
4. Dorset’s egg medium (Mycobacteria)
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a) Blood agar b) Chocolate agar C) Loeffler’s Serum Agar

30. • Is a media used to increase the relative number of desired
microorganisms.
• i.e. favor heavy growth of a certain class of m.o.
• Nutrients and incubation conditions are manipulated in such a way that
the growth of a desired class of microbe in encouraged, or the growth
of undesired microbes is inhibited.
Example: Tetrathionate Broth (Salmonella/ Shigella)
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Tetrathionate Broth
Enrichment broth

31. Selective Media
• A media that contains a substance that inhibits the growth of some
microbes while allowing the growth of others. i.e. they select for
certain microbes.
Example:
1. Lowenstein-Jensen (Mycobacterium
tuberculosis)
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2. Sabouraud’s Dextrose Agar
(for cultivation of fungi)
3. Cetrimide Agar (Pseudomonas)

32. Differential or Indicator Media
• These media distinguish one type of mos. from another that is
growing on it in the presence of indicator.
• The indicator reacts differently to different organisms and
produce morphologically distinguishable colonies
Example:
1. Litmus Milk 2. Urea Agar Medium
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Litmus Milk Urea Agar

33. Both selective & differential.
• Used both to distinguish colonies of a desired organism and inhibit the
growth of other microbes.
Example:
1. Mannitol Salt Agar (MSA)
2. Eosin Methylene Blue Agar (EMB)
3. Deoxycholate Citrate Agar (DCA)
4. Thiosulfate Citrate Bile Salts Medium (TCBS)
5. Triple Sugar Iron Agar (TSI)
6. Bismuth Sulfite Agar (BS)
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TSI
BS

34. Media for anaerobes
• These media supply little or no dissolved oxygen on which anaerobes
can thus grow.
• Contains “Reducing Agents” that are autooxidizable with great affinity
to oxygen (glutathione, cysteine, thioglycollate, ascorbic acid).
• Contains a low concentration of agar which increases the viscosity of the
medium and decreases the rate of diffusion of oxygen.
• Bacteria grow in the medium according to their respiratory
requirements.
• Media filled in deep columns, tubes, (anaerobic mos. grow in the
bottom)
Example:
• Fluid thioglycollate medium
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Fluid
Thioglycollate
Medium
Resazurin
layer
Fluid
Thioglycollate
Medium
Resazurin
Layer
Redox
indicator dye

35. 2.3. Pure culture techniques
• Pure culture is a population of cells arising from a single cell
• can be accomplished from mixtures by a variety of procedures,
including streak, spread and pour plates.
• Colonies are macroscopically visible growths or clusters of
microorganisms on solid media.
• Colony growth is most rapid at the colony's edge because oxygen
and nutrients are more available; growth is slowest at the colony's
center.
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36. Streak Plate Method

37. Streak Plate Method

38. Spread vs pour plates

39. • Microbiologists use five basic techniques to manipulate, grow,
examine, and characterize microorganisms in the laboratory:
1. Inoculation:- Introduce a tiny sample (the inoculums) into a
container of nutrient medium
2. Incubation:- Incubator creates proper growth temperature to
promote multiplication of microbes
3. Isolation:- Separating a single bacterial cell from other cells
4. Inspection, Growth is observed
5. Identification:- What type of microbe is it?
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40. • Colony morphology helps microbiologists identify bacteria
because individual species often form colonies of characteristic
size and appearance
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41. • Once mos. are isolated in pure culture it is necessary to keep
the culture alive.
• Cultures can be stored
• At 40C in the refrigerator.
• By slant, petridishes etc.
• In liquid nitrogen at -780C
• Deep freezers at -70 to -120 0C
• Lyophilization or freeze drying ( frozen, dehydration and
sealed under vacuum)
• Change ice directly to water vapor.
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42. 2.4. Staining techniques
• 1. Bacteria on slide
• 2. Air Dry
• 3. Bacteria are heat fixed to the slide
• 4. Stain is applied
Staining Reaction
• Dyes/ Stains - salts composed of a positive and negative ion, one
of which is colored (chromophore)
• Basic Dyes - chromophore is the positive ion
• It reacts with negatively charged molecule
• dye+ Cl-
• Acid Dyes - chromophore is the negative ion
• It reacts with positively charged molecule.
• dye- Na+
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43. • Bacteria are slightly negative, so are attracted to the positive
chromophore of the Basic dye
• Common Basic Dyes
• crystal violet
• methylene blue
• safranin
• basic fuchsin
• Acid Dyes - used for Negative Staining (background is stained)
• Mordant - intensifies the stain or coats a structure to make it thicker
and easier to see after it is stained
Example:
• Flagella - can not normally be seen, but a mordant can be
used to increase the diameter of the flagella before it is
stained. Salmonella
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44. 1. Simple staining
• Bacteria have nearly the same refractive index as water, therefore,
when they are observed under a microscope they are opaque or
nearly invisible to the naked eye.
• Different types of staining methods are used to make the cells and
their internal structures more visible under the light microscope.
• The cell Simple stains use one dye that stains the cell wall.
• s are then visible against a light background.
• Shows morphology
• Size
• Shape
• Arrangement
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45. Steps:
1. Place the slide on the staining rack.
2. Flood the slide with a basic stain: either crystal violet (1 min.),
Safranin (2 min.), or Methylene blue (2 min.).
3. Wash the stain off the slide with deionized water.
4. Blot the slide with bibulous paper.
5. Observe under microscope.
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46. A/ Gram Stain
• 1884 Hans Christian Gram
• most important stain used in Bacteriology
• Divides all Bacteria into 2 groups:
• Gram (+)
• Gram (-)
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1. Crystal violet
2. Differential Stains
• Distinguish Bacterial groups and Specific Structures
• React differently with different types of bacteria
2 Most Common differential stains
1. Gram Stain
2. Acid-Fast Stain

47. 2. Grams Iodine (mordant)
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3. Alcohol

48. 4. Safranin (Counterstain)
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Results
• Gram (+) Purple
• Gram (-) Red
• Difference - due to structure of cell
wall
– Gram (+) Thick cell wall
– Gram (-) Thin cell wall

49. 49
B/ Acid - Fast Stain
• Differential Stain – divides bacteria into 2 groups
 Acid – Fast
 Non Acid – Fast
• Used to identify organisms in the Genera Mycobacterium (high lipid
and wax content in cell wall)
• The most common method for acid fast stain is Ziehl Neelsen stain
 Acid - Fast Cells Red
 Non Acid - Fast Blue

50. 3. Special Stains
A/ Capsule Stain B/ Flagella Stain
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Klebsiella pneumoniae Spirillum volutans
C/ Endospore Stain
Bacillus cereus

51. Gram Staining