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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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