Simple staining

Microbiology LAB #5!!!

Simple Staining

[pic 1]

Dr. Kari Murad

The College of Saint Rose

Bright field microscopy

• Image of the specimen is made from light that is transmitted from the light source to the ocular.

– Specimen restricts light transmission

– Background permits light transmission

**REVIEW: parts of a compound light microscope & resolution of a compound light microscope

Parts of a microscope

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How it works…

Object        Object

[pic 3]

Ocular

Condenser w/ Objective Lens Lens diaphragm

Resolution

[pic 4]

[pic 5]

[pic 6]

Light vs. EM

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Bacterial Shapes

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[pic 9]The microscopes in this lab cost approximately $2000 EACH-AVOID breaking one!!

• ALL BACKPACKS in CUPBOARDS!

• CARRY WITH TWO HANDS, CLOSE to your BODY.

• LOCATION/ LOCATION… excess cord stored, cover stored… all away from bunsen burner flame!

• Always START on Low Power Objective - focus! Adjust Light Diaphragm.

• MOVE to 10x – focus! Adjust Light Diaphragm.

• MOVE to 40x – focus! Adjust Light Diaphragm.

• OIL IMMERSION Lens – know how to use correctly – focus (fine ONLY)! Adjust Light Diaphragm.

• DRAW

• END on Low Power Objective and wipe of OIL IMMERSION lens!

Staining Vocab

• Stains- solutions consisting of a solvent (usually water or ethanol) and dye.

• Chromophore- the charged region of a dye molecule that gives it color and interacts with the cell being stained (chromophores can be positively charged (basic) or negatively charged (acidic).

(60 sec)

Simple Stains (Basic stains)

• Create a contrast between bacteria and the background

Bacterial cell is stained

Background is not stained

[pic 10]

Apply basic stain

(positive charge C+)

Bacterial cell

(negative charge due to cell wall)

Simple staining protocol: see page 100

Simple stains: Safranin (30 sec), Crystal Violet (30 sec), Carbol fuchsin (15 sec), Methylene Blue

Negative Stains (Acidic stains)

• Create a contrast between bacteria and the background

[pic 11]

Bacterial cell is not stained

Background is stained

[pic 12]

Apply acidic stain

(negative charge C-)

Bacterial cell

(negative charge

due to cell wall)

...