1、Getting Ready for Todays LabGrab your lab check-in card and the dirty copies of Lab#2 and#3 that we saved from last week.Grab your plastic box of supplies.Put on your lab coat and have your goggles,lab card and dirty copies of lab exercises/reports on the bench next to you.Plug in and turn on your m
2、icroincinerator.TURN IN:No lab report or Pre-lab due this week.End of Todays LabAfter you have cleaned your lab bench,copy your Lab#2 and#3 material on to a clean copy to take home.I must see you throw out your dirty copy of Lab#2.You may turn in your dirty copy of Lab#3 to save for use next week.La
3、b Report#2 and#3 due next week,as week,as well as Pre-lab#4.Laboratory 3Differential StainingsueGThree Main Things We are Doing TodayI.Looking at Results from Last Weeks CulturesII.Learning to use an Inoculation Loop for Streak Plate MethodIII.Differential StainsI.Looking at Results from Last Weeks
4、CulturesOther Types of Media:McConkeys,Blood Agar&Mannitol SaltLook at the plates on your lab bench that are red/pink In color.Blood Agar=very dark redMcConkeys=lighter,purplish-pinkMannitol Salt-=orangish-pinkMacConkeys(already prepared)MacConkeys is both a selective&differential media.1.What Does
5、Selective Mean?MacConkeys is selective media because it inhibits the growth of some organisms Gram positive bacteria.2.What Does Differential Mean?Because“neutral red”has been added to the media,it is also differential.-Bacteria that use lactose(a type of sugar)for food,produce acidic metabolites wh
6、ich causes the pH indicator neutral red to turn red.-These“lactose fermenters”will grow in red colonies while non-lactose fermenters will be colorless and clear.Members of the family Enterobacteriaceae(Gram negative bacilli)are the most frequently encountered bacteria isolated from many types of cli
7、nical specimens.They are most commonly lactose fermenters.Blood agar Most specimens received in a clinical microbiology lab plated onto Blood Agar,since it supports the growth of most organisms.Blood agar contains 5%sheep blood.This media is differential because:Certain bacteria produce enzymes(hemo
8、lysinshemo-lice-ins)that act on the red cells to produce either:Beta hemolysis:Enzymes lyse the blood cells completely,producing a clear area around the colony.Alpha hemolysis:Incomplete hemolysis produces a greenish discoloration around the colony.Gamma hemolysis:No effect on the red cells.Blood ag
9、ar is usually inoculated from a patients throat swab.Microbiologist try to detect presence of Group A beta hemolytic Streptococci(a Gram-positive cocci that causes Beta hemolysis on blood agar.)Major human pathogen in this group is Streptococcus pyogenes,the causative agent of strep throat.Normal th
10、roat flora will exhibit alpha or gamma hemolysis.Beta means strep.Mannitol SaltIs a selective media with a high concentration of NaCI(7.5%).Most bacteria cannot survive in this environment.The genus Staphylococcus,however,grows well in this media.Mannitol Salt is also differential because it contain
11、s a dye that identifies organisms that ferment mannitol.The organic acids produced change the dye from red to yellow.This medium works well for identifying pathogenic staphylococci,such as Staphylococcus aureus,which will ferment mannitol.Most non-pathogenic staphylococci will not ferment mannitol.R
12、ecording Results from Last LabCultures1.Record results of environmental samples,for each quadrants 1 4(TSY and MacConkeys).a.Are MacConkey colonies pink or colorless?b.What does it mean if they are pink?2.Describe results from touch plates and arm plates(TSY).a.What is the difference between plates
13、1,2 and 3?3.Record results from throat swab plate(Blood agar).a.Do you see Alpha hemolysis?Beta hemolysis?Gamma hemolysis?b.Which hemolysis pattern is bad news?4.Record results from nasal swab plates(TSY and Mannitol Salt).a.Did any of the colonies turn the Mannitol Salt medium yellow?b.What would i
14、t mean if they did?II.Inoculation Loop&Streak Plate MethodWe will culture our unknown onto MacConkeys and Mannitol Salt.To do a streak plate technique,we will need to use an innoculation loop.AKA smear loop,inoculation wand or microstreaker Simple tool used to retrieve an inoculum from a culture of
15、microorganisms.Always sterilize in a flame until it becomes red hot before and after each use.By doing this,the same tool can be reused in different experiments without fear of cross-contamination.Be sure that your inoculation loop has cooled before using it to retrieve an inoculum or streak a plate
16、!If you hear the medium sizzle the loops too hot!MicroincineratorStreak Plate TechniqueWhen we want to isolate a specific species of bacteria,we need to use a technique that will spread out the original“parent bacteria”in a sparse enough pattern so that after growth,individual colonies will result.A
17、fter incubation,the 4th quadrant of your plate should have dots.These small“dots”are individual colonies,and represent millions of bacteria of the same speciesIII.Differential StainsDifferential StainsMost stains used in microbiology are differential stains.Use more than one dye so that different ce
18、lls,chemicals or structures can be distinguished.Preparing Specimens for Viewing Differential StainsGram StainDistinguishes between two large groups of microorganisms:-purple staining,Gram-positive cells-pink staining,Gram-negative cellsThese two types of cells differ significantly in the chemical a
19、nd physical structure of their cell wall.The structure of the thinner cell walls of Gram negative bacteria cannot hold the dyes previously used,once the decolorizer is applied.Crystal violet(1 min):rinse:Iodine(1 min):rinse:Acetone Alcohol(1015 sec):rinse:Safrinin(1 min)Mordant is a chemical used to
20、 fix the staining reactionsueGPreparing Specimens for Viewing Differential StainsAcid-Fast StainThis stains the cells of the genera Mycobacterium and Nocardia,which cause many diseases in humans,including tuberculosis,leprosy,and other lung and skin infections.Cells of these bacteria have large amou
21、nts of waxy lipid in their cells walls,so the Gram stain and other water-based stains dont work well on them.Cells are determined to be acid-fast or not,based on whether they retain their primary stain after decolorization.Create a smear of the organism that you are testing.Cover the smear with a st
22、rip of blotting paper.Saturate paper with Ziehls carbolfuchsin and heat for 3 5 minutes.Remove blotting paper.Wash slide with tap water and then decolorize the smear for 10-15 seconds with acid alcohol.Rinse again with tap water.Apply crystal violet for 1 minute,wash,blot dry.OBSERVATION OF MICROORG
23、ANISMSBlotting paper:Ziehls carbolfuchsin(3 5 min heat):rinse:Acid Alcohol(10 15 sec):rinse:crystal violet(1 min)musPreparing Specimens for Viewing Differential StainsEndospore StainSome bacteria produce endospores,dormant,highly-resistant cells inside the cytoplasm of the bacteria,that can survive
24、environmental extremes(desiccation,heat,harmful chemicals)Most notable genera:Bacillus and ClostridiumEndospores cannot be stained by normal staining procedures because their walls are practically impermeable to all chemicals.Endospore stain uses heat to drive the primary stain,malachite green into the endospore.After cooling,the sample is decolorized with water and counter stained with safranin.Results in green stained endospores and red-colored vegetative cells.Malachite Green(5 min heat):rinse:safrinin(20 sec)bweE
