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
• Indole
• Methyl Red
• Voges-Proskauer
• Citrate 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 (NO3 to NO2)
• 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
Top Related