E ndospores staining Microbial culture media and sterilization Methods of inoculation and isolation of pure culture Asst Lec Mariam A Ali AlMustaqbal University college 2 ID: 1026215
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1. Medical microbiologyLab 4: Endospores staining; Microbial culture media and sterilization; Methods of inoculation and isolation of pure culture.Asst. Lec. Mariam A. AliAl-Mustaqbal University college 2 st Class, 1 st Semester Department of pharmacy
2. Bacterial Spores (endospore)The Vegetative Cell Gives Rise to One Spore Endospore is dormant stage of some bacterium that allows it to survive under unfavorable conditions that would normally be lethal such as extreme drought or heat The endospore is able to survive for long periods of time until environmental conditions again become favorable for growth. The endospore then germinates, producing a single vegetative bacterium.
3. Not all bacterial species can form sporesA few genera of bacteria produce endospore such as Clostridium (gangrene) and Bacillus (anthrax), both of them are gram + rodsEndospore production is associated with Gram Positive bacteriaSince not all bacteria form endospores, we can use this as an identification factor3
4. The shape of the spore is an identifying characteristicSwelled vs. Not swelled4Bacterial cellsporeBacterial cellspore
5. The location of the spore is also an identifying characteristicCentral, Sub-Terminal, and Terminal spores5
6. Some spore forming bacteria are capable of causing diseaseClostridium botulinum – botulismClostridium perfingens – gas gangreneClostridium tetani – tetanusBacillus anthracis – Woolsorter’s Disease and wound infectionsThe Schaeffer-Fulton Stain Procedure is used to differentiate between endospores and vegetative cells6
7. Schaeffer-Fulton Stain Procedure1. Make a smear. Air Dry. Heat fix2. Flood the smear with Malachite Green stain3. Cover the flooded smear with a square of filter paper4. Steam slide for 10 minutes (every minute, add a few more drops of Malachite Green stain)5. Allow slide to cool (after the 10 min. steam process)7
8. Schaeffer-Fulton Stain Procedure6. Drain slide and rinse for 30 seconds with DI water (discard filter paper).7. Put slide on steam rack.8. Flood smear with Safranin (counter stain). This stains the vegetative cell. (Leave for 1 minute) 9. Drain the slide and rinse with DI water.10. Blot Dry.11. Use oil immersion objective to view.8
9. 9Special stain: endospore staining (malachite green)
10. Media: Providing Nutrients in the LaboratoryAt least 500 different typesContained in test tubes, flasks, or Petri dishesInoculated by loops, needles, pipettes, and swabsSterile technique necessaryClassification of mediaPhysical stateChemical compositionFunctional type
11. Classification of Media by Physical StateA- Liquid mediaB- Semisolid mediaC- Solid mediaby Chemical ContentA-Synthetic mediaB- non syntheticby FunctionA- General purpose media B-Enriched MediaC- Selective MediaD-Differential Media
12. 1- Classification of Media by Physical StateA- Liquid media: water-based solutions, do not solidify at temperatures, ex: pepton water, meat infusion broth.Growth seen as cloudiness or particulatesB- Semisolid media: clotlike consistency at room temperatureUsed to determine motility and to localize reactions at a specific siteC- Solid media: a firm surface on which cells can form discrete coloniesLiquefiable and nonliquefiableUseful for isolating and culturing bacteria and fungiEx: nutrient agar, blood agar.Semi solid media solid media broth media
13. 2- Classification of Media by Chemical ContentA-Synthetic media: compositions are precisely chemically definedB-Complex (nonsynthetic) media: if even just one component is not chemically definable
14. 3- Classification of Media by FunctionA- General purpose media: to grow as broad a spectrum of microbes as possibleUsually nonsyntheticContain a mixture of nutrients to support a variety of microbesExamples: nutrient agar and broth, brain-heart infusion, trypticase soy agar (TSA).
15. B-Enriched MediaEnriched media- contain complex organic substances (for example blood, serum, growth factors) to support the growth of fastidious bacteria. Examples: blood agar: a differential media to distinguishes between types of hemolysis bacteria A- Complete Beta hemolysis bacteria Streptococcus pyogenes , Streptococcus agalactiaeB- Partial Alpha hemolysis bacteria Streptococcus pneumonia , Streptococcus viridansC- Non hemolytic bacteria Streptococcus bovis , Streptococcus faecalis
16. C- Selective MediaSelective media- contains one or more agents that inhibit the growth of certain microbes but not others. Example: Mannitol salt agar (MSA):a differential media to distinguishes between Staphylococcus aureus and other staph. spp.Staphylococcus aureus can fermented mannitol and convert the phenol red of mannitol to yellow
17. D-Differential media- allow multiple types of microorganisms to grow but display visible differences among those microorganisms. Ex: MacConkey agar: a differential media to distinguishes between lactose fermenting bacteria which appeared red ex: E. coli, Klebsiella, Enterobacter aerogenes and non lactose fermenting bacteria which appeared a colorless. Ex Proteus, Shigella, and Salmonella.
18. FORMS OF CULTURE MEDIA1. Broth tube: are tubes containing a liquid medium. A typical nutrient containing broth medium such as Trypticase Soy broth , nutrient broth After incubation, growth (development of many cells from a few cells) may be observed as one or a combination of three forms: Pellicle: A mass of organisms is floating on top of the broth.b. Turbidity: The organisms appear as a general cloudiness throughout the broth .c. Sediment: A mass of organisms appears as a deposit at the bottom of the tube.
19. 2. Slant tubes: are tubes containing a nutrient medium plus a solidifying agent, agar-agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface .3. Stab tubes (deeps): are tubes of hardened agar medium which are inoculated by "stabbing" the inoculum into the agar .
20. Agar Plate Culture of a Bacterium4- Agar plates: are sterile petri plates that are aseptically filled with a melted sterile agar medium and allowed to solidify. Plates are much less confining than slants and stabs and are commonly used in the culturing, separating, and counting of microorganisms.
21. Three common forms of agar media.Inoculation of agar slants and deeps.
22. A medium is sterilized (living organisms removed) before usage in the lab with different sterilization methods include:Autoclaving, Dry-heat, Filtration, UV exposure, Ethylene oxide.
23. Autoclave:The principle of sterilization in an autoclave is that steam under pressure is used to produce a temperature of 121ºC which if held for 15 minutes all microorganisms including bacterial endospores will be destroyed.Sterilization of equipment and materials Wire loop: Heat to redness in Bunsen burner flame. Empty glassware and glass (not plastic!) pipettes and Petri dishes: Either, hot air oven, wrapped in either grease proof paper or aluminum and held at 160-180ºC for 2 hours. Culture media and solutions: Autoclave/pressure cooker. Glass spreaders and metal forceps: Flaming in alcohol.
24. Inoculation and IsolationInoculation: producing a cultureIntroduce a tiny sample (the inoculums) into a container of nutrient medium Isolation: separating one species from anotherSeparating a single bacterial cell from other cells and providing it space on a nutrient surface will allow that cell to grow in to a mound of cells (a colony). If formed from a single cell, the colony contains cells from just that species.
25. Figure 3.2
26. Streak Plate MethodStreak plate method- small droplet of culture or sample spread over surface of the medium with an inoculating loopUses a pattern that thins out the sample and separates the cellsFigure 3.3 a,b
27. Loop Dilation MethodLoop dilation, or pour plate, method- sample inoculated serially in to a series of liquid agar tubes to dilute the number of cells in each successive tubesTubes are then poured in to sterile Petri dishes and allowed to solidifyFigure 3.3 c,d
28. Spread Plate MethodSpread plate method- small volume of liquid, diluted sample pipette on to surface of the medium and spread around evenly by a sterile spreading toolFigure 3.3 e,f