Two -methods are generally used, (wet method) and (dry and fix method (.for Studying Microbes with a Compound Microscope

Wet Method. There are two primary methods generally used for studying microorganisms in wet conditions

wet mount method

hanging drop method.

The wet mount

A drop of fluid containing microorganisms.

The fluid spreads out in a thin layer between coverslip and slide.

The hanging Drop Method

It is used to observe the motility, germination, or fission of microorganisms.

Dry and fix method

Microorganisms, particularly bacteria, being too small need their permanent preparation be made by drying and fixing them on clean slide with or without staining.

For preparing a dry mount:

A drop of distilled water with a small amount of culture is spread as a thin smear on a clean slide. The smear is allowed to dry and it is then 'fixed' by passing it through a flame two to three times with the smeared slide away from the flame. If desired, this dried and fixed amount may be stained and the preparation dried again for observation under the microscope.

Bacterial Shapes :

Bacteria have three basic shapes:

Round cells (Cocci) , Rod-shaped cells are bacilli , spiral-shaped cells

Types of stainTypes of stain

ChromophoreChromophore– Positive: basic stainPositive: basic stain

Attaches to cell wall and bacterium appears Attaches to cell wall and bacterium appears coloredcolored

Direct StainDirect Stain

– Negative: acidic stainNegative: acidic stainRepelled from cell wall; stains backgroundRepelled from cell wall; stains background

Negative StainNegative Stain

Simple stainsSimple stains– To observe basic external structures of cell To observe basic external structures of cell

with brightfield scope (cellular morphology)with brightfield scope (cellular morphology)– Reagent: Methylene BlueReagent: Methylene Blue

SmearsSmears

Heat FixingHeat Fixing– KillKill– Stops autolysisStops autolysis– Adherence to slideAdherence to slide

Air dry first to prevent lysis (boiling)Air dry first to prevent lysis (boiling)

Use of a single basic dye is called a simple Use of a single basic dye is called a simple stain.stain.

A mordant may be used to hold the stain or A mordant may be used to hold the stain or coat the specimen to enlarge it.coat the specimen to enlarge it.

The Gram stain classifies bacteria into gram-The Gram stain classifies bacteria into gram-positive and gram-negative.positive and gram-negative.

Gram-positive bacteria tend to be killed by Gram-positive bacteria tend to be killed by penicillin and detergents.penicillin and detergents.

Gram-negative bacteria are more resistant to Gram-negative bacteria are more resistant to antibiotics.antibiotics.

Gram StainGram Stain

PurposePurpose– To view cellular morphologyTo view cellular morphology– Diagnostic purposes: What type of cell wall a Diagnostic purposes: What type of cell wall a

bacterium hasbacterium has

Gram PositiveGram Positive– Thicker peptidoglycan layer and no outer Thicker peptidoglycan layer and no outer

membranemembrane

Gram NegativeGram Negative– Thinner Peptidoglycan and an out membraneThinner Peptidoglycan and an out membrane

Gram Type Cell WallsGram Type Cell Walls

Reagents for Gram StainReagents for Gram StainCrystal Violet (purple)Crystal Violet (purple)– Primary stain; positive stainPrimary stain; positive stain– Stains cell wall purpleStains cell wall purple

IodineIodine– MordantMordant– Combines with CV to form an insoluble complex that gets trapped in Combines with CV to form an insoluble complex that gets trapped in

thicker peptidoglycan layersthicker peptidoglycan layers– What happens if you skip this step?What happens if you skip this step?

EthanolEthanol– DecolorizerDecolorizer– CV-I complex washed out of Gram negative organisms because it CV-I complex washed out of Gram negative organisms because it

cannot be trapped by peptidoglycan layer; flows right through outer cannot be trapped by peptidoglycan layer; flows right through outer membranemembrane

Safranin (pink)Safranin (pink)– CounterstainCounterstain– Simple positive stain that provides contrasting dye for decolorized cells Simple positive stain that provides contrasting dye for decolorized cells

(Gram negative)(Gram negative)– Stains all cells, but only the negative ones actually appear pink.Stains all cells, but only the negative ones actually appear pink.

Color of Color of

Gram + Gram + cellscells

Color ofColor of

Gram – Gram – cellscells

Primary stain:Primary stain:

Crystal violetCrystal violet

PurplePurplePurplePurple

Mordant:Mordant:

IodineIodine

PurplePurplePurplePurple

Decolorizing agent:Decolorizing agent:

Alcohol-acetoneAlcohol-acetone

PurplePurpleColorlessColorless

Counterstain:Counterstain:

SafraninSafranin

PurplePurpleRedRed

Thick Thick peptidoglycanpeptidoglycan

90% peptidoglycan90% peptidoglycan

Teichoic acidsTeichoic acids

1 layer1 layer

Not many Not many polysaccharidespolysaccharides

In acid-fast cells, In acid-fast cells, contains mycolic contains mycolic acidacid

Gram-positive cell wallsGram-positive cell walls Gram-negative cell Gram-negative cell wallswalls

Thin peptidoglycanThin peptidoglycan

5-10% peptidoglycan5-10% peptidoglycan

No teichoic acidsNo teichoic acids

3 layers3 layers

Outer membrane has Outer membrane has lipids, polysaccharideslipids, polysaccharides

No acid- fast cells No acid- fast cells (mycolic acid)(mycolic acid)

The characteristic compound found in all true bacterial cell walls is peptidoglycan. The amount of PPG is among one of the differences between the GP and GN cell walls.

The crucial step in the staining process is the decolorizing step. The most accepted theory about the rationale for the Gram staining process is the one proposed by Salton.

This theory relies on the fact that the PPG is found in layers and the stain molecules are trapped within the many layers of the GP CW when they form the complex with the mordant Iodine molecules. Since the GN CWs lack much PPG the amount of stain captured in those CWs is much lesser.

When the cells are treated with the decolorizer – the ethanol – this causes denaturation of the proteins in the outer membrane of the GN CWs resulting in gaping holes in these CWs that lead to the removal of the crystal violet-iodine complexes easily, leaving these cells unstained.

The counterstain -safranin- thus is used to make these cells visible.

There are 4 conditions to be followed for a valid Gram staining procedure: 

Young cultures - must be young within 18-24hrs old (older cultures lose their Gram staining properties due to changes in the CWs as the cells get older)

Thin smearthicker or uneven smears will result in uneven stainingand decolorization

Fresh reagents - of proper strengthControl cultures - for a known GP bacterium and GN culture

(S.aureus & E.coli)