Laboratory Biochemical Assays Mcb1010 L
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Laboratory Biochemical Assays Mcb1010 L

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Laboratory Biochemical Assays Mcb1010 L Laboratory Biochemical Assays Mcb1010 L Presentation Transcript

  • Laboratory Biochemical Assays A Quick & Concise Review
  • Parts of the Microscope
    • Coarse Focus
    • Fine Focus
    • Stage
    • Condenser
    • Objectives
    • Oculars
    • Light Source
  • Negative Staining
    • Natural size & shape
    • No heat fix; prevents distortion
    • Congo Red
    • HCl
  • Negative Stain
    • Bacteria will be clear cells
    • Background will be stained dark or navy blue
  • Gram Stain
    • Hallmark staining procedure
    • Gram positive vs. gram negative
    • Crystal Violet
    • Grams Iodine
    • 95% ethanol
    • Safranin
  • Gram Stain
    • Purple are gram +
    • Pink are gram -
  • Acid Fast Stain
    • Mycobacterium ssp.
    • Thick & waxy, lipoidal cell wall
    • Carbol fuschin
    • Acid alcohol
    • Brilliant green
  • Acid Fast Stain
    • Acid fast cells will be pink
    • Non-acid fast cells will be green
  • Spore Stain
    • Bacillus & Clostridium ssp.
    • Malachite green w/ heat
    • Safranin
  • Spore Stain
    • Spores are green
    • Vegetative cells are pink
  • Capsule Stain
    • Klebsiella ssp.
    • No heat fixing
    • Crystal violet
    • Copper sulfate
  • Capsule Stain
    • Capsule will appear as a light blue, or clear halo around the bacterial cell
  • Differential & Selective Media
    • Selective:
    • Isolate specific groups of bacteria
    • Chemical substances inhibit the growth of one type of bacteria
    • Permit the growth of another
  • Differential & Selective Media
    • Differential:
    • Distinguish between morphologically& biochemically related groups of bacteria
    • Produce characteristic change in the surrounding media or their appearance on the media
  • Mannitol Salt Agar (MSA)
    • High salt (7.5%)
    • Phenol red
    • Mannitol
  • Mannitol Salt Agar (MSA)
    • Halophiles
    • Mannitol fermentation
    • Useful in isolating SA
  • SA on MSA
  • Blood Agar
    • Fastidious organisms
    • Hemolytic properties
  • Blood Agar
    • Gamma hemolysis – no hemolysis (SA)
    • Alpha hemolysis – incomplete lysis; greenish halo
    • Beta hemolysis – complete hemolysis; clear halo (SP)
  • SP on Blood Agar
  • MacConkey Agar
    • Crystal Violet
    • Bile Salts
    • Lactose
    • Neutral Red
  • MacConkey Agar
    • Inhibits gram positive organisms
    • Ability to ferment lactose
    • EC vs EA
    • Salmonella vs. Shigella
  • EA & EC on MacConkey Agar
  • Eosin Methylene Blue Agar (EMB)
    • Lactose vs. non-lactose fermenters
    • EC vs. EA
  • Eosin Methylene Blue Agar (EMB)
    • EC has a metallic green sheen to it’s growth
    • EA has a fish-eye appearance
  • EA vs. EC on EMB Agar
  • Starch Hydrolysis
    • Exoenzymes amylase & maltase
    • Breakdown starch in the agar
  • Starch Hydrolysis
    • Plate turns blue-black after the addition of iodine?
    • Plate shows a clear zone around the bacterial colony after the addition of iodine?
  • Starch Hydrolysis
  • Gelatin Tube
    • Exoenzyme gelatinase
    • Protein hydrolysis
  • Gelatin Tube
    • Gelatin is liquified?
    • How to confirm?
    • If it remains a liquid?
    • If it re-solidifys?
  • Carbohydrate Fermentation
    • Can bacteria ferment sugars?
    • Acid?
    • Acid & Gas?
    • 3 tubes, lactose, dextrose and sucrose
    • Phenol red
    • Durham Tube
  • Carbohydrate Fermentation
    • Yellow tube?
    • Bubble in the Durham tube?
    • Red or pink tube? What happened?
  • Carbohydrate Fermentation
  • Triple Sugar Iron (TSI)
    • Enterobacteriaceae
    • Glucose (0.1%)
    • Alkaline slant/acid butt
    • Acid slant/acid butt
    • Alkaline slant/alkaline butt
    • Blackening of the butt
    • Read the tube when?
  • Triple Sugar Iron (TSI)
  • IMViC Test
    • I ndole
    • M ethyl Red
    • V oges-Proskauer
    • C itrate Utilization
  • Indole
    • Tryptophanase degrades tryptophan
    • Indole is a waste product of the reaction
  • Indole
    • Kovacs reagent
    • Cherry-red reagent layer if indole is present
  • Indole
  • Methyl Red
    • Ferment glucose in the MRVP broth
    • Large quantities of acid end-products
  • Methyl Red
    • Methyl Red indicator used to detect pH changes (reacts at a pH~4)
    • Red color in the broth indicates acid end-products and is a positive test
    • EC MR+
    • EA MR-
  • Voges-Proskauer
    • Glucose fermentation
    • Acid end-products
    • Conversion of these acid end-products to non-acid end-products
  • Voges-Proskauer
    • Barrits Reagents A & B
    • Detects acetoin, a non-acidic end-product
    • Rose-colored band at the top of the broth
    • EC VP-
    • EA VP+
  • MRVP Test
  • Citrate Utilization
    • Citrate as a sole carbon source
    • Simmons citrate agar slant
    • Bromthymol blue – pH indicator
  • Citrate Utilization
    • Blue slant – positive test
    • Green slant – negative test
  • Citrate Utilization
  • Litmus Milk
    • Skim milk
    • Lactose
    • Milk proteins – casein, lacto-albumin
    • pH indicator is none other than litmus
  • Litmus Milk
    • Lactose fermentation - pink
    • Litmus reduction - white
    • Gas formation – cracks/fissures
    • Curd formation – acid vs. rennet
    • Proteolysis – purple/brown, whey-like
    • Alkaline reaction – unchanged or deep purple
  • Litmus Milk
  • Litmus Milk
  • Nitrate Reduction Test
    • Nitrates to Nitrites (NO 3 to NO 2 )
    • Anaerobic Process
    • Nitrate Broth
    • Sulfanilic Acid (Solution A)
    • Alpha-napthylamine (Solution B)
  • Nitrate Reduction Test
    • Mix solutions A & B
    • Red color develops?
    • No color develops? Significance?
    • Add Zinc Powder?
    • Color?
    • No color?
  • Nitrate Test
  • Catalase
    • Degrade H2O2 using catalase
    • Phenomenon of aerobic bacteria
    • TSA
    • H2O2
  • Catalase
    • Bubbles?
    • No bubbles?
  • Catalase
  • Oxidase
    • Cytochrome oxidase C activity
    • Phenomenon of aerobic respiration
    • TSA plate
    • p-aminodimethylaniline
    • Artificial electron donor
  • Oxidase
    • Black color develops?
    • No color or pink coloration?
  • Oxidase, at the bottom