Lab #3 Preparation of Smears and Simple Stain

You are performing a simple stain. What information about your organism(s) will you be able to collect using this procedure?

-cell size -cell shape -arrangement of cells

The preparation of a bacterial smear can be difficult to learn. List four reasons why this process ‭may be problematic for some.

1. If you don't completely air dry your slide, you will end up boiling your microorganisms when heat-fixing. 2. When mixing bacteria with water, if you over mix it can result in inaccurate cell patterns. 3. When heat-fixing, if you hold the slide over the flame too long, you will incinerate the cells. 4. If you don't kill the bacteria during heat-fixation, their ability to accept stains is reduced and you will not see anything under the microscope.

How to stain cells

1. obtain small portion of culture 2. smear on slide (if not broth, add a drop of water before smearing) 3. air dry 4. heat fix by very briefly passing the slide over the flame 3x

Colored anion

Acidic stain

Before heat fixation, a wet smear of cells on a slide must first be ________.

Air-dried

Colored cation

Basic Stain

What are the limitations of a simple stain?

Can only determine cell shape and size.

Failure to heat fix slides will most likely result in _________.‭

Cells not sticking to the slide

Why do we stain cells?

Individual cells or specific cellular structures have little contrast with the clear background of the slide. Colors increases the contrast and makes them easier to view.

Simple stain examples

Methylene blue, safranin, Carbolfuchsin, Crystal Violet

Simple stains use ________ and differential stains use ________.

One type of dye ; More than one type of dye

Which of the organisms you observed has a nucleus?

Saccharomyces cerevisiae

Staining kills bacterial cells.

True

If you are preparing a smear from solid media what must be added to the slide before adding the cells?

Water

What are the two reasons why smears are heat-fixed?

alter the cells to enable better uptake of stain adhere the cells to the slide

If you overmix smear

can lead to inaccurate bacterial cell patterns (chains may appear as clumps)

Colored ion in stains

chromophore

If too many bacteria on smear

clear differentiation of individual cells will be difficult

Simple stains cannot distinguish

differences between cell components b/c all bacteria take up the stain

If too few bacteria on smear

have to spend long time looking for a cell to observe

Differential stains can differentiate

organisms' cellular components

Stains are

salts in which one of the ionic components is colored

You will perform this procedure using Escherichia coli, a prokaryotic organism, and Saccharomyces cerevisiae, a eukaryotic organism. What differences do you predict you will observe between these two organism?

size and shape