Lab Quiz #1 (Lab 1 -> Enteric Organism Identification + Enterotube)

Why do we perform stains?

To create contrast between bacteria cell & background. Bacteria cells are clear in appearance.

Types of Stains

• Simple Stain

• Differential stain

Simple Stain

• uses a single dye to visualize cells

• information we get from it is morphology and presence/absence of cells

• Examples from lab: Methylene Blue stain

Basic dyes

Upon ionization, the chromogen has a positive charge

Acidic dyes

Upon ionization, the chromogen has a negative charge

Simple Stain with Methylene Blue

• Cell has an overall negative charge + Methylene blue is a positively charged dye = stains the cell; cell shape becomes apparent

• Sticks to cells, appears colored on white background

If you were to use a negative dye (nigrosin) to perform a simple stain, how do you think that stain will work?

• The negatively charged molecules of the dye will be attracted to the positively charged background.

• Repels negatively charged cells

• White cells on black background

Nigrosin dye

• Acidic and negatively charged

• Repelled by negatively charged cells

• White cells on black background

Nigrosin Stain Steps

1. Place a drop of nigrosin at one end of the slide

2. Place a loopful of the inoculum into the drop of stain and mix with the loop.

3. Place a slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the edge of the applied slide.

4. Push the slide away from the previously spread drop of suspended organisms, forming a gradient along the slide.

NOTE: No rinsing involved - does not require heat fixation

Why does simple staining with methylene blue require heat fixation?

Bacteria needs to stick to the slide after stain rinses.

Disadvantages of heat fixing

• Cells are dead; can’t see motility

• Cell shape may get distorted due to heat

Advantage of using nigrosin over positively charged dye for simple stain

• No heat fixing required

• Undistorted by heat

Smear Prep Steps

1. Add loopful of water to each wax circle

2. Sterilize loop and pick up tiny amount of cells from slant. Place cells in water in circle #1 and spread around.

3. Transfer loopful from circle #1 to #2. Spread around

4. Allow to dry COMPLETELY. Heat fix by passing in front of incinerator 4-5 times

5. Apply stain, sit for 60 seconds. Rinse with water and plot dry.

Methylene Blue Stain Steps

1. Make 2 thick wax circles on your slide

2. Add a LOOP of water to each circle

3. Touch edge of loop to cell growth on slant, spread cells in first circle

4. Immediately transfer a loopful of circle 1 to circle 2

5. Allow to dry COMPLETELY

6. Heat fix by passing slide in front of incinerator 4-5 times

7. Apply Methylene blue for 60 seconds

8. Rinse with water and blot dry

Annotations on Microscopy Pictures

• Cell name

• Morphology

• Color

• Total magnification (specify if oil immersion was used)

cocci

spherical in shape (must be perfect circles)

diplo-

pair

strepto-

chain

staphylo-

cluster

tetrad

packet of 4

sarcina

packet of 8

bacilli

rod-shaped

coccobacilli

intermediate shape between cocci and bacilli

Resolution

Ability of microscope to distinguish two adjacent objects as separate

Resolving Power

λ/2NA

Decrease wavelength (λ)

Use blue light filter

Increase NA

Use highest objective on microscope

Total Magnification

Magnification of eyepiece (10x) multiplied by magnification of the objective

Viewing cells under microscope

• Focus on wax line at 400X

• Move lens and add oil. Switch to 1000X, make minor adjustments to refocus

• Do not use oil on other objectives

Pure Culture

Only 1 strain of bacteria living in the culture

What is/are the advantage(s) of obtaining a pure culture?

• Easy identification of microorganism

• Isolation of specific strains

• Diagnostic purposes

How might you go about attempting to obtain a pure culture?

Perform a Quadrant Streak

Escherichia coli

• Cell shape: bacillus

• Gram-negative

Serratia marcescens

• Cell shape: bacillus

• Gram-negative

Staphylococcus aureus

• Cell shape: staphylococcus

• Gram-positive

Bacillus megaterium

• Cell shape: bacillus

• Gram-positive

Staphylococcus epidermidis

• Cell shape: staphylococcus

• Gram-positive

Differential stain

uses 2 dyes to distinguish between cells based on physiological features or to differentially stain different cellular structures

Gram Stain

• Differentiates cells based on cell wall structure

• Primary stain: crystal violet

• Mordant: iodine

• Decolorizing agent: ethanol

• Counterstain: safranin

Endospore Stain

• Differentiates between vegetative cells and endospores

• Primary stain: malachite green

• Decolorizing agent: water

• Counterstain: safranin

Primary stain (ES)

Apply malachite green stain to smear and heat over steam bath

• Vegetative cell

  • Malachite green is a basic stain (positively charged)

  • Penetrates negatively-charged cells

• Endospore

  • Without heat, the stain is unable to penetrate the spore coat

  • With heat, spore coat opens to allow stain in

Decolorizing (ES)

Remove from heat and wash smear with water

• Vegetative cell

  • malachite green is water-soluble

  • water easily washes stain from vegetative cells

• Endospore

  • When heat is removed, the spore coat closes back up, trapping stain inside the spore

Secondary stain (ES)

Apply safranin to smear

• Vegetative cell

  • Decolorized cells will be stained by the safranin (basic, positively charged stain)

  • Appear red/pink under microscope

• Endospore

  • Endospores won’t absorb secondary stain

  • Will appear green under microscope

Endospore Stain Steps

1. Prepare heat-fixed smear from slant

2. Apply malachite green

3. Suspend slide over steam bath for five minutes

4. Wash with water until water runs clear

5. Apply safranin for one minute

6. Wash with water until water runs clear

7. Blot dry

Gram Stain Steps

1. Prepare 2 heat-fixed smears (E. coli + S. epidermidis)

2. Stain with crystal violet (1 minute)

3. Wash with water until water runs clear

4. Stain with Gram’s iodine (1 minute)

5. Wash with ethanol for a couple of seconds, just until the moment the ethanol runs clear

6. Rinse with water for about 2 seconds to stop decolorization

7. Blot dry if needed. Stain with safranin for 1 minute

8. Wash with water until water runs clear. Blot slide dry.

Colony Morphology: Forms

• Circular

• Irregular

• Rhizoid

Colony Morphology: Margins

• Entire

• Lobate

• Undulate

• Serrate

• Filamentous

Colony Morphology: Elevation

• Flat

• Raised

• Convex

• Umbonate

Complex Media

• media supplemented with nutritious ingredients such as yeast extract

• supports growth of a wide range of organisms

• Ex: nutrient broth/agar used for quadrant streak exercise

Selective Media

media used to select for the growth of specific types of bacteria

Differential Media

media used to differentiate between bacteria based on different physiological characteristics

Mannitol Salt Agar (MSA) Plate

Selective:

• selects for salt-tolerant microorganisms

  • such as skin microorganisms

• Key ingredient: 7.5% NaCl limits growth of non-tolerant organisms

Differential:

• Differential for mannitol fermentation

• Key ingredient: mannitol added as a carbon source; phenol red added as pH indicator

Result Interpretation:

• Growth?

  • Yes = salt-tolerant organism

  • No = not salt-tolerant

• Color?

  • Yellow = mannitol fermented

  • Red = mannitol not fermented

  • How?

    • Fermentation produces organic acids that drop the pH levels, turning phenol red to yellow.

Eosin Methylene Blue (EMB) Plate

Selective:

• selects for Gram-negative bacteria

• Key ingredients: eosin and methylene blue dyes are inhibitory towards Gram-positive bacteria; only Gram-negative organisms can grow in their presence

Differential:

• Differential for lactose fermentation

• Key ingredients: lactose added as carbon source; organic acid produced from fermentation causes dyes to precipitate out of media and onto surface of cells

Result Interpretation

• Growth?

  • Yes = Gram-negative bacteria

  • No = Gram-positive bacteria

• Color?

  • Colorless/Lilac = no lactose fermentation

  • Dark Purple = + for lactose fermentation

  • Metallic Green = ++ for lactose fermentation

  • How?

    • Drop in pH from organic acids (created by fermentation) causes dyes in media to precipitate out onto surface of cells

Phenol Red CHO broth test

• Tests for fermentation of glucose

  • Can test for fermentation of different sugars depending on what carbon source is added

• Glucose fermentation can also produce gases (H₂ and CO₂)

  • Durham tube is added to catch any gas formed

• Result interpretation:

  • Yellow = + for glucose fermentation

    • Gas in durham tube if gas was produced

  • Red = - for glucose fermentation

  • How?

    • Organic acid lowers pH; red → yellow

Voges-Proskauer (VP) test

• Tests for the production of 2,3-butanediol

• Result interpretation:

  • Red = + for 2,3-butanediol production

  • Not red = - for 2,3-butanediol production

  • How?

    • Adding Barritt’s reagents reacts with the acetoin (precursor to 2,3-butanediol) to form red-colored product

Kligler’s Agar Deep Slant

• Tests for cysteine desulfurase activity

• Also tests for fermentation of glucose and lactose

• Result interpretation:

  • Black = + for precipitate formation

  • Not black = - (no precipitate formed)

  • How?

    • H₂S + Fe = black precipitate

  • Glucose and Lactose Fermentation results

    • Yellow all throughout = glucose and lactose fermented

      • space at the bottom = gas production

    • Yellow bottom red top = only glucose fermented

    • Red = no fermentation occurred

Indole Production test

• Tests for tryptophanase activity

• Result interpretation:

  • Red = + for tryptophanase activity

  • Not red = - for tryptophanase activity

  • How?

    • Kovac’s reagent reacts with indole (formed by tryptophanase) to form red-colored product at the surface of tube

Phenylalanine deaminase slant test

• Tests for phenylalanine deaminase activity

• Result interpretation:

  • Green = + for phenylalanine deaminase activity

  • Not green = - for phenylalanine deaminase activity

  • How?

    • Added ferric chloride reacts with phenylpyruvic acid to form green colored product

Motility Test

• Tests for cell motility

• Result interpretation

• + = pink all over

• - = pink in stab line only

• How?

  • Actively growing cells reduce TTC

    • TTC (oxidized = colorless) → TTC (reduced = pink)

Simmon’s Citrate test

• Tests for utilization of citrate as sole carbon source for growth

  • Depends on presence of citrate transporter (citrate permease)

• Media contains

  • carbon source: citrate

  • pH indicator: bromothymol blue

• Result interpretation

  • + = blue

  • - = green

  • How?

    • Bromothymol blue turns blue when pH goes up because of Na₂CO₃ production

Urease test

• Tests for urease activity

• Result interpretation

  • + = hot pink

  • - = yellow

  • How?

    • Phenol red turns pink when pH rises because of NH₃ production

Enterotube II Multitest System

• single tube containing 12 compartments each with a different media and an inoculating needle

• The needle is used to touch a single isolated colony and then is drawn through the compartments in order to inoculate all of the media

• This allows you to simultaneously perform multiple standard biochemical tests for identification of members of Enterobacteriaceae.