Downloaded 164 times
Uploaded byHuang Yu-Wen
Module 3a culture and staining
This document discusses the nutritional and environmental requirements for bacterial growth, including the need for carbon, nitrogen, phosphorus, and other elements. It describes different types of culture media like liquid broth, semi-solid agar, and solid agar plates and how they are used. The document also covers bacterial colony morphology, describing smooth, mucoid, and rough colony types. It discusses factors that provide a suitable growth environment for bacteria, such as oxygen levels, temperature, pH, and osmotic pressure requirements.
More Related Content
Similar to Module 3a culture and staining
Module 3a culture and staining
- 1. Bacterial Staining &Culture Stains & Types of Dyes Cultivating Microorganisms Microbiology - HTL
- 2.
- 3. What Organisms needto 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.
- 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 aculture 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.
- 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.
- 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.
- 21.
- 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 mucoidcolonies 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 roughcolonies s Granulated in appearance and hard to emulsify in NSS s Eg. Corynebacterium diptheriae Microbiology - HTL 2
- 27. Possible descriptions ofbacterial 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 suitableenvironment 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.
- 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.
- 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.
- 36.
- 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.
- 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 microrganismsdie 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 ofStaining 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.
- 45.
- 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.
- 48.