Module 3a - Culture and Staining
-
Upload
maebelene-melo -
Category
Documents
-
view
241 -
download
0
Transcript of Module 3a - Culture and Staining
![Page 1: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/1.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 1/48
Microbiology - HTL 1
Bacterial Staining & Culture
Stains & Types of Dyes
Cultivating Microorganisms
![Page 2: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/2.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 2/48
Microbiology - HTL 2
![Page 3: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/3.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 3/48
Microbiology - HTL 3
What Organisms need to grow
Nutrition
Carbon
Oxygen Nitrogen
Phosphorus
Sulfur Trace elements
Organic growth factors
![Page 4: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/4.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 4/48
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
![Page 5: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/5.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 5/48
Microbiology - HTL 5
More chemicals:
Other elements:
• Potassium
• Magnesium
• Calcium
Used as cofactors
for enzymes
Trace elements
• Iron
• Copper
• Molybdenum
• Zinc
![Page 6: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/6.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 6/48
Microbiology - HTL 6
![Page 7: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/7.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 7/48Microbiology - HTL 7
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
![Page 8: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/8.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 8/48Microbiology - HTL 8
Media preparation
materials
weigh out
dissolve in solvents filter to clarify
adjust pH
place in containers sterilize & control
store & refrigerate
![Page 9: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/9.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 9/48Microbiology - HTL 9
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
![Page 10: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/10.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 10/48Microbiology - HTL 10
![Page 11: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/11.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 11/48Microbiology - HTL 11
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
![Page 12: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/12.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 12/48Microbiology - HTL 12
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.
![Page 13: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/13.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 13/48Microbiology - HTL 13
![Page 14: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/14.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 14/48
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
![Page 15: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/15.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 15/48
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
![Page 16: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/16.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 16/48
![Page 17: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/17.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 17/48
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)
![Page 18: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/18.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 18/48
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
![Page 19: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/19.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 19/48
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
![Page 20: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/20.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 20/48
Microbiology - HTL 20
![Page 21: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/21.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 21/48
Microbiology - HTL 21
![Page 22: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/22.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 22/48
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
![Page 23: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/23.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 23/48
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
![Page 24: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/24.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 24/48
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 )
![Page 25: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/25.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 25/48
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
![Page 26: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/26.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 26/48
Microbiology - HTL 26
R or rough colonies
Granulated in appearance and hard to emulsify in NSS
Eg. Corynebacterium diptheriae
![Page 27: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/27.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 27/48
Microbiology - HTL 27
Possible descriptions of bacterial
growth on agar slants:
Arborescent- branched
Beaded
Echinulate – pointed Filiform – even
Rhizoid – rootlike
Spreading
![Page 28: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/28.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 28/48
O & T
![Page 29: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/29.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 29/48
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
![Page 30: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/30.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 30/48
Microbiology - HTL 30
H H d t ti & O ti
![Page 31: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/31.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 31/48
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
![Page 32: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/32.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 32/48
Microbiology - HTL 32
Osmotic pressure:
Osmoprotectants – concentration of solutes > solvent
• High conc Plasmolysis • Low conc Plasmoptosis
Halophiles – high salt concentration
atmospheres - barotolerant
![Page 33: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/33.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 33/48
Microbiology - HTL 33
M i N b f
![Page 34: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/34.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 34/48
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
![Page 35: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/35.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 35/48
Microbiology - HTL 35
![Page 36: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/36.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 36/48
Microbiology - HTL 36
![Page 37: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/37.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 37/48
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
![Page 38: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/38.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 38/48
Microbiology - HTL 38
![Page 39: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/39.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 39/48
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
![Page 40: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/40.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 40/48
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
![Page 41: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/41.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 41/48
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
![Page 42: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/42.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 42/48
Microbiology - HTL 42
Bacterial relationships
Free living
Symbiosis
Commensalism Parasitism
Synergism
Antagonism
![Page 43: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/43.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 43/48
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
![Page 44: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/44.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 44/48
Microbiology - HTL 44
![Page 45: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/45.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 45/48
Microbiology - HTL 45
![Page 46: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/46.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 46/48
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
![Page 47: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/47.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 47/48
Microbiology - HTL 47
![Page 48: Module 3a - Culture and Staining](https://reader030.fdocuments.in/reader030/viewer/2022021112/577ce7b51a28abf103959f2f/html5/thumbnails/48.jpg)
7/31/2019 Module 3a - Culture and Staining
http://slidepdf.com/reader/full/module-3a-culture-and-staining 48/48