Experiment 2: Simple Staining + Aseptic Technique

Aseptic Technique: 2 reasons its important

1. Prevents the microbe working with from contaminating the samples or work area or person

2. Prevents microbes in the air from contaminating bacteria being worked with

How does aseptic technique prevent contamination?

dry heat sterilization (high temo and no moisture) to eliminate all microbes

what does sterile mean?

free of life

non sterile

anything you you can see and touch in the lab

what is the goal of aseptic technique?

keep cultures pure while working with them

pure culture

collection of cells of a single strain or species growing in media free of contamination or signs of life

Work area aseptic technique

use bench disinfectant

neatly organize workspace to prevent spills or hazards

using bunsen burners to be aseptic

dry heat sterilization

two cones in bunsen flame: outer hot and inner blue cooler (hottest part is the tip of blue)

flame creates sterile field due to hot air and updraft (umbrella) that pushes contaminants away

have it lit within 1 foot radius of flame

have a BLUE flame not orange `

flaming inoculating loop/needle

hold with dom hand and point down, insert at 45 deg angle at hottest part of the flame until red

allow it to cool

how to aseptically remove broth cultures (4)

1. hold agitated tube in non dom hand and loop in dom hand

2. remove cap of tube with dom pinky and put the tip through the flame

3. put cooled down loop into the tube, take some inoculum

4. flame mouth of tube again and close tube

what is agar

gelatinous substance

made of carbs, galactan extracted from marine algae of Geledium

creates solid media for growing microorganisms

when does agar turn into a solution/melt

when heated to nearly 100 deg C and remains a liquid until 43 deg C

but once solidied, it has to be reheated to 100 to make it liquid again

what does solid agar allow for

colony morphology, purity, growth patterns to be studied

how to inoculate the plate or grab solid culture

never take the lid off the plate completely (open at angle)

flame loop/needle (aseptically) and take some inocolum onto the loop/needle

why is it difficult to see bact cells when observed with convential light microscopes

bc theyre transparent

what is simple staining

uses a single type of stain/dye to create a contrast between cells and the background (all cells are gonna be he same colour)

advantage of simple staining

allows us to see cell morphology (shape)

most used dyes for simple staining (3)

methylene blue - blue

fuchsin - red

crystal violet - purple

Types of staining procedures (2)

positive and negative

direct positive staining procedures

basic stain has a positivly charged (cationic) chromophore

what is a chromophore

part of a molecule responsible for its colour

generates colour of the stain

why do bacteria absorb these stains

bacteria have a net negative charge on their outer surface to create an attraction to the cationic dye so direct binding

methylene blue has a ___ chromophore

blue; methylene blue+ chloride- which gives a positive blue stained organism

basic stain and most stains are applied to bacterial smears which are?

heat fixed; bacteria is killed and adheres to the slide so it doesn’t move when stained/washed

coagulates the cytoplasmic proteins to make them more visible

exp 2 learning objectives (5)

1. keep stuff sterile

2. know that microbes are everywhere

3. aseptically use broth cultures

4. simple staining methods

5. identify cell morphologies and arragments

exp 2 materials

1. loefflers methlyne blue stain

2. pure broth: Staphylococcus epidermis

3. pure broth of Prestia megaterium

agitate b4 using broth

you know there is growth in broth cultures if you see ______ in tubes?

turbidity/cloudiness

how to make a smear/slide

1. draw circle and label

2. agitate, aseptically take some incolum and put on slide in the circle (dime sized sample) - (second loopful if culture is too diluted)

3. let smears air dry (DONT hold near flame to dry, might distort shape)

4. when dry, heat fix by passing slide through the flame 2-3 times smear side up

what does heat fixing do?

coagulates the bacterial proteins and fixes them on the slide

how to stain (after heat fixing)

1. use 1-3 drops of methylen blue dye and leave stain on for two mins

2. dont let it dry out (add some more if need be)

3. pour excess off and rinse with some water from squirt bottle until water coming from slide is colourless

4. remove excess water by BLOTTING dry w paper towel, dont rub

5. examine cell morphology/arrangement under microscope

Prestia megaterium

streptobacillus, 1000x TM

Staphylococcus epidermis

staphylococcus, 1000x TM

benefit and limitation of simple staining

benefit: see morphology shape/size/ arrangement by increasing contrast

limitation: unable to differentiate between types of cells or bacteria bc et same colour