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