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1. Bacterial Staining & Culture
Stains & Types of Dyes
Cultivating Microorganisms
Microbiology - HTL

2. Microbiology - HTL

3. What Organisms need to grow
s Nutrition
s Carbon
s Oxygen
s Nitrogen
s Phosphorus
s Sulfur
s Trace elements
s Organic growth
factors
Microbiology - HTL

4. Chemical requirements:
s Carbon – structural backbone of living matter
s Nitrogen – form the amino group of the amino
acids of proteins
• Nitrogen fixation – Cyanobacteria, Rhizobium
s Sulfur – Synthesize sulfur containing amino
acids and vitamins.
• Eg. Thiamine and Biotin
s Phosphorus – synthesis of Nucleic acids and
the phospholipids of the cell membranes
Microbiology - HTL

5. More chemicals:
s Other elements: s Trace elements
• Potassium • Iron
• Magnesium • Copper
• Calcium • Molybdenum
• Zinc
s Used as cofactors
for enzymes
Microbiology - HTL

6. Microbiology - HTL

7. Culture Media
s Anything that possesses nutritional and
environmental requirements for bacterial
growth
s Culture -- is a group of organisms obtained in
a culture media
s 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
Microbiology - HTL

8. Media preparation
s materials
s weigh out
s dissolve in solvents
s filter to clarify
s adjust pH
s place in containers
s sterilize & control
s store & refrigerate
Microbiology - HTL

9. Ingredients of a culture media:
s protein
s nitrogen
s carbohydrate
s solidifying agents /agar & gelatin
• Agar – polysaccharide extracts of seaweeds and
are most commonly used as base medium
s chemical substance
s dyes & indicators
s enriching substance – e.g. chocolate, blood,
glycerine, egg, albumin
Microbiology - HTL

10. Microbiology - HTL 1

11. Types of media (consistency)
s Liquid / broth - motility, transport , enrichment,
biochemical tests
• Eg. Thioglycolate broth, BHI, TSB, Nutrient broth
s semi-solid - 0.25% agar + liquid
motility, anaerobic culture, stock culture,
microaerophiles, & biochemical tests
• SIM, Fletcher
s solid - 1-2% agar
used for studying colonies
• BAP, CAP, MSA, EMB, TSI, SCA
Microbiology - HTL 1

12. Types of Culture – ( specie )
s Pure culture – made up of only one
pure specie
s Mixed culture – made up of organisms
belonging to different specie
s Stock Culture – pure culture of
organism used as a source of supply for
industry, research or academic uses.
Microbiology - HTL 1

13. Microbiology - HTL 1

14. Types of media – (composition)
s Synthetic Culture Medium – exact
composition is known or ingredients are
known
s Non-synthetic – exact composition is
not known
s Tissue culture medium – used for
culturing living cells
• Eg. Human cancer cell lines
Microbiology - HTL 1

15. Type of media – (method of
dispensing or distributed)
s Plated Medium – dispensed in
petridishes
s Tube medium – dispensed in test tubes
• Slant
• Butt
• Butt/slant
Microbiology - HTL 1

16. Types of Media – (B ased on use)
s Simple medium – supports the growth of
fastidious microorganisms
• Used for routine cultivation and maintenance
of microorganisms
– Eg. Nutrient broth, Nutrient agar
s Enrichment medium – containing nutritive
suplements needed for some microbes to
growth
– Eg. Peptone water – growth of V. cholera
Microbiology - HTL 1

17. Continued…
s 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)
Microbiology - HTL 1

18. More…
s Differential Medium – distinguishes organism
growing together by differences in their
cultural characteristic
• Eg. EMB, MCA, MSA, TCBS (Trypticase Citratr
Bile Salt Agar ), SSA
s Selective Medium – promotes growth of
desirable organism but at the same time
inhibiting the growth of others.
• Used for culture of specific organism
Microbiology - HTL 1

19. Last na…
s 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
Microbiology - HTL 1

20. Microbiology - HTL 2

21. Microbiology - HTL 2

22. Types of media ( function)
s Defined - Glucose
s Complex - egg, blood, beef, yeast, milk
s Selective - SPS agar (Clostridium)
s Differential - Blood agar
s Selective/differential - MacConkey agar
Crystal violet / lactose
s Enrichment - Nitrogen free media
Microbiology - HTL 2

23. Bacterial Colony:
s Colonies – groups of bacteria forming
on certain solid media as a result of
several divisions of one or several
specific type of organism
s Only one type of bacteria will be found
in a bacterial colony
Microbiology - HTL 2

24. Types of colonies:
s 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 )
Microbiology - HTL 2

25. M or mucoid colonies
s Associated with capsulated & virulent
organisms
s 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
Microbiology - HTL 2

26. R or rough colonies
s Granulated in appearance and hard to
emulsify in NSS
s Eg. Corynebacterium diptheriae
Microbiology - HTL 2

27. Possible descriptions of bacterial
growth on agar slants:
s Arborescent- branched
s Beaded
s Echinulate – pointed
s Filiform – even
s Rhizoid – rootlike
s Spreading
Microbiology - HTL 2

28. Providing a suitable environment
s Oxygen Requirement
s Temperature
s pH
s Osmotic Pressure
s Hydrostatic Pressure
s Salt Concentration
Microbiology - HTL 2

29. Oxygen & Temperature
requirements :
s Obligate aerobes s Thermophilic
s Facultative aerobe • above 50
s Obligate anaerobe s Mesophilic
s Facultative • best at 37
anaerobe s Psychrophiles
s Microaerophiles • below 5
s Capnophiles s MGT, mGT , OGT
s Hyperthermophiles
Microbiology - HTL 2

30. Microbiology - HTL 3

31. pH, Hydrostatic & Osmotic
pressure
s pH scale 1-14 s Buffers: chemicals
• Acidophiles that is used to
• Alkaliphiles neutralize the acids
and maintain the
s wide range but proper pH
internally usually
neutral 6.5-7.5
s Methods
• colorimetric
• electrometric
Microbiology - HTL 3

32. Osmotic pressure:
s Osmoprotectants – concentration of
solutes > solvent
• High conc  Plasmolysis
• Low conc  Plasmoptosis
s Halophiles
– high salt concentration
s atmospheres - barotolerant
Microbiology - HTL 3

33. Microbiology - HTL 3

34. Measuring Numbers of
Microorganisms
s direct microscopic
• Petroff Hauser counter
s electronic count
s plate count – Standard agar Plate
s MPN – Most probable number
s viable count – Trypan blue
s Filtration -
s Turbidity – indirect way of extimating
s dry weight – for filamentous organisms e.g.
molds
s metabolic activity – reduction Test e.g. oxygen
uptake
Microbiology - HTL 3

35. Microbiology - HTL 3

36. Microbiology - HTL 3

37. Growth of Microorganisms
s Population - microbial growth
s Doubling time/Generation Time
• Time interval until the completion of
next bacterial division
s Growthrate
s Exponential growth
Microbiology - HTL 3

38. Microbiology - HTL 3

39. Bacterial Growth Curve
s Latent phase ( Lag 8000
phase) 7000
6000
s Logarithmic phase 5000
(Log phase) 4000
3000
s Stationary phase 2000
1000
s Death Phase 0
(Phase of Decline)
Microbiology - HTL 3

40. The way microrganisms die
s Rate of microbial death
- temperature, type of microbe,
physiologic state, presence of other
substances that might protect
s Decimal reduction time - D value
(time in minutes -- 90% population)
s Thermal death point - TDP
s Thermal death time - TDT
Microbiology - HTL 4

41. Bacterial Death
s Death is due to
• Lack of food
• Accumulation of toxins & dead debris
• Development of unfavorable conditions
s Death is the complete ceasation of
multiplication
Microbiology - HTL 4

42. Bacterial relationships
s Free living
s Symbiosis
s Commensalism
s Parasitism
s Synergism
s Antagonism
Microbiology - HTL 4

43. Common Types of Staining
s Simple Stain s Types of Dyes:
• methylene blue • Basic -Safranin ,
carbol fuchsin,
s Differential stain Methylene blue
• Gram’s Stain • Acidic - Eosin,
• Acid Fast Stain acid fuchsin,
congo red
s Special Stain
s Mordants
• Wirtz Conklin
s Decolorizers
• Leifson
Microbiology - HTL 4

44. Microbiology - HTL 4

45. Microbiology - HTL 4

46. Gram Stain & Acid Fast Stain
s Crystal Violet s Carbol Fuchsin
s Gram’s Iodine s Heat
s 95% Alcohol s Acid Alcohol
s Safranin s Methylene Blue
• (+) Violet to • (+) Pink to Red
Purple • (-) Blue to Violet
• (-) Pink to Red
Microbiology - HTL 4

47. Microbiology - HTL 4

48. Microbiology - HTL 4