The document discusses the nutritional requirements, growth conditions, and culturing techniques for microorganisms. It describes the essential chemical elements like carbon, nitrogen, phosphorus and trace elements that microbes need to grow. It also explains the different types of culture media used to cultivate microorganisms under varying oxygen levels, temperatures, pH and osmotic pressures. Various methods to measure microbial growth and induce bacterial death are also summarized. Common staining techniques to identify bacteria including Gram stain and acid-fast stain are outlined in the concluding paragraphs.

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