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Microbiology - HTL 1
Bacterial Staining & Culture
Stains & Types of Dyes
Cultivating Microorganisms
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Microbiology - HTL 2
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Microbiology - HTL 3
What Organisms need to grow
Nutrition
Carbon
Oxygen Nitrogen
Phosphorus
Sulfur Trace elements
Organic growth factors
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Microbiology - HTL 4
Chemical requirements:
Carbon – structural backbone of living matter
Nitrogen – form the amino group of the amino acids of proteins • Nitrogen fixation – Cyanobacteria, Rhizobium
Sulfur – Synthesize sulfur containing amino acids and vitamins.• Eg. Thiamine and Biotin
Phosphorus – synthesis of Nucleic acids and the phospholipids of the cell membranes
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Microbiology - HTL 5
More chemicals:
Other elements:
• Potassium
• Magnesium
• Calcium
Used as cofactors
for enzymes
Trace elements
• Iron
• Copper
• Molybdenum
• Zinc
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Microbiology - HTL 6
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Culture Media
Anything that possesses nutritional and environmental requirements for bacterial growth
Culture -- is a group of organisms obtained in a culture media
Colony – is a culture containing group of
bacteria forming on a solid culture medium as a result of separated division of 1 or a few organisms
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Media preparation
materials
weigh out
dissolve in solvents filter to clarify
adjust pH
place in containers sterilize & control
store & refrigerate
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Ingredients of a culture media:
protein
nitrogen
carbohydrate
solidifying agents /agar & gelatin • Agar – polysaccharide extracts of seaweeds and
are most commonly used as base medium
chemical substance dyes & indicators
enriching substance – e.g. chocolate, blood,glycerine, egg, albumin
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Types of media (consistency)
Liquid / broth - motility, transport , enrichment,biochemical tests • Eg. Thioglycolate broth, BHI, TSB, Nutrient broth
semi-solid - 0.25% agar + liquid motility, anaerobic culture, stock culture,microaerophiles, & biochemical tests • SIM, Fletcher
solid - 1-2% agar used for studying colonies
• BAP, CAP, MSA, EMB, TSI, SCA
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Types of Culture – ( specie )
Pure culture – made up of only one pure specie
Mixed culture – made up of organisms belonging to different specie
Stock Culture – pure culture of
organism used as a source of supply for industry, research or academic uses.
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Microbiology - HTL 14
Types of media – (composition)
Synthetic Culture Medium – exact composition is known or ingredients are known
Non-synthetic – exact composition is not known
Tissue culture medium – used for
culturing living cells • Eg. Human cancer cell lines
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Microbiology - HTL 15
Type of media – (method of
dispensing or distributed)
Plated Medium – dispensed in petridishes
Tube medium – dispensed in test tubes • Slant
• Butt
•
Butt/slant
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Microbiology - HTL 17
Continued…
Enriched medium – containing nutritive supplements for growth of some
microorganisms • Eg. BAP – contains Factor V ( Coenzyme –
heat labile factor Nicotinamide dinucleotide) & Factor X (Hemin heat
stable factor)
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Microbiology - HTL 18
More…
Differential Medium – distinguishes organism growing together by differences in their cultural characteristic
• Eg. EMB, MCA, MSA, TCBS (Trypticase Citratr Bile Salt Agar ), SSA
Selective Medium – promotes growth of
desirable organism but at the same time inhibiting the growth of others.
• Used for culture of specific organism
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Microbiology - HTL 19
Last na…
Special/Specific culture medium • Same as the purpose of selective culture
medium
• Used to isolate hard to isolate or grow strains
• Eg. Petrognani, Lowenstein, Petroffs – for
Mycobacterium tuberculosis • Thayer Martin medium – Neisseria
• McBride Agar – Listeria monocytogenes
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Microbiology - HTL 20
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Microbiology - HTL 21
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Microbiology - HTL 22
Types of media ( function)
Defined - Glucose
Complex - egg, blood, beef, yeast, milk
Selective - SPS agar (Clostridium) Differential - Blood agar
Selective/differential - MacConkey agar
Crystal violet / lactose Enrichment - Nitrogen free media
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Microbiology - HTL 23
Bacterial Colony:
Colonies – groups of bacteria forming on certain solid media as a result of
several divisions of one or several specific type of organism
Only one type of bacteria will be found
in a bacterial colony
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Microbiology - HTL 24
Types of colonies:
S or smooth colonies
• Uniform texture and homogenecity
•
Forms glistening texture • Easily emulsified in Normal saline solution
• Usually associated with virulent organisms
•
Eg. Gram negative organisms ( Neisseria )
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Microbiology - HTL 25
M or mucoid colonies
Associated with capsulated & virulent organisms
Exhibits slimy or watery confluent appearance and are positive to string test which indicates presence of Gm –
organisms like K. pneumoniae in EMB agar – demonstrated with 3% KOH
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Microbiology - HTL 26
R or rough colonies
Granulated in appearance and hard to emulsify in NSS
Eg. Corynebacterium diptheriae
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Microbiology - HTL 27
Possible descriptions of bacterial
growth on agar slants:
Arborescent- branched
Beaded
Echinulate – pointed Filiform – even
Rhizoid – rootlike
Spreading
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O & T
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Microbiology - HTL 29
Oxygen & Temperature
requirements :
Obligate aerobes
Facultative aerobe
Obligate anaerobe Facultative
anaerobe
Microaerophiles Capnophiles
Thermophilic
• above 50
Mesophilic • best at 37
Psychrophiles
• below 5 MGT, mGT , OGT
Hyperthermophiles
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Microbiology - HTL 30
H H d t ti & O ti
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Microbiology - HTL 31
pH, Hydrostatic & Osmotic
pressure
pH scale 1-14
• Acidophiles
•Alkaliphiles
wide range but internally usually
neutral 6.5-7.5 Methods
• colorimetric
•
electrometric
Buffers: chemicals that is used to neutralize the acids and maintain the proper pH
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Microbiology - HTL 32
Osmotic pressure:
Osmoprotectants – concentration of solutes > solvent
• High conc Plasmolysis • Low conc Plasmoptosis
Halophiles – high salt concentration
atmospheres - barotolerant
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Microbiology - HTL 33
M i N b f
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Microbiology - HTL 34
Measuring Numbers of
Microorganisms
direct microscopic • Petroff Hauser counter
electronic count
plate count –
Standard agar Plate MPN – Most probable number viable count – Trypan blue Filtration -
Turbidity –
indirect way of extimating dry weight – for filamentous organisms e.g.
molds metabolic activity – reduction Test e.g. oxygen
uptake
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Microbiology - HTL 35
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Microbiology - HTL 36
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Microbiology - HTL 37
Growth of Microorganisms
Population - microbial growth
Doubling time/Generation Time
• Time interval until the completion of next bacterial division
Growth rate
Exponential growth
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Microbiology - HTL 38
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Microbiology - HTL 39
Bacterial Growth Curve
Latent phase ( Lag phase)
Logarithmic phase (Log phase)
Stationary phase
Death Phase (Phase of Decline )
0
1000
2000
3000
40005000
6000
7000
8000
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Microbiology - HTL 40
The way microrganisms die
Rate of microbial death - temperature, type of microbe,
physiologic state, presence of other substances that might protect
Decimal reduction time - D value
(time in minutes -- 90% population) Thermal death point - TDP
Thermal death time - TDT
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Microbiology - HTL 41
Bacterial Death
Death is due to
• Lack of food
•
Accumulation of toxins & dead debris • Development of unfavorable conditions
Death is the complete ceasation of
multiplication
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Microbiology - HTL 42
Bacterial relationships
Free living
Symbiosis
Commensalism Parasitism
Synergism
Antagonism
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Microbiology - HTL 43
Common Types of Staining
Simple Stain
• methylene blue
Differential stain
• Gram’s Stain
• Acid Fast Stain
Special Stain
• Wirtz Conklin
• Leifson
• Negative
Types of Dyes:
• Basic -Safranin ,carbol fuchsin,
Methylene blue • Acidic - Eosin,
acid fuchsin,
congo red Mordants
Decolorizers
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Microbiology - HTL 44
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Microbiology - HTL 45
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Microbiology - HTL 46
Gram Stain & Acid Fast Stain
Crystal Violet
Gram’s Iodine
95% Alcohol Safranin
• (+) Violet to
Purple • (-) Pink to Red
Carbol Fuchsin
Heat
Acid Alcohol Methylene Blue
• (+) Pink to Red
•(-) Blue to Violet
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Microbiology - HTL 47
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