Pure Culture - PowerPoint Presentation

Slide1

Pure Culture

Pure Culture Techniques

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Turn on Incinerators and hot plates (low heat)

**Slide2

Microbial Culture

Method of multiplying microbial organisms by letting them reproduce in a predetermined culture media under controlled laboratory conditionsSlide3

Types of Media

Agar: jelly like substance derived from seaweed; thickening agent

We use agar because most microorganisms cannot digest agar so it provides a firm surface on which to grow and we can pick out individual coloniesBroth: liquid media (same as agar without the thickening agent) Slide4

Types of Agar

We can add specific ingredients to agar to grow or inhibit specific microorganisms

Selective: inhibit or help growth of certain organisms with the use of specific chemicalsDifferential: organisms produce characteristic changes or growth patterns dependent on the ingredients present

Supportive

: supports the growth of most organisms

Enrichment

: supplemented with

highly

nutritious materials that allow for the growth of fastidious (picky) organismsSlide5

Types of Agar

Tryptic Soy Agar (TSA) : Supportive media; general media used to grow most microorgansimsSlide6

Types of Agar

Eosin Methylene Blue (EMB): Selective and DifferentialSelects for Gram negative organisms (inhibits the growth of Gram Positive

organisms)Has Eosin Y and Methylene Blue- indicator dyes that react with any acidic products resulting from lactose fermentation to color the colonies

Lactose fermentation causes precipitation of the dyes on the surface of the colonies resulting in different colors-

Differential

Large amounts of acid → green metallic sheen

Small amounts of acid → pink

No fermentation → colorlessSlide7

Pure Cultures

Needed to identify bacteria and for antibiotic sensitivityCan be achieved by:

Pour platesIsolation StreaksSlide8

Pure Culture

Pour Plate: Serial dilution of the original sample is performed and a small amount of the final dilution is added to melted agar. The melted agar is poured into an empty sterile plate. Colonies will develop subsurface. Slide9

Pure Culture

Streak Plate

: the original culture is directly diluted across an agar surface using an inoculating loop

The

main idea

of a pure culture is

to dilute or thin out the original sample until the organism of interest is isolated and pure. Slide10

Making a Streak Plate Slide11

What do we need?

Split up into groups of 2 1 TSA plate/group

Mix 1 (Room Temp)1 EMB plate/group

Mix 2 (37°)

LABEL YOUR PLATES

Name/Initials

Class Section

Which mix you used Slide12

Exercise 6

Smear Preparation and Simple Staining Slide13

Smear Preparation

The way a smear is prepared will determine how well your stain will come out3 goals to a good smear

Making sure the cells adhere to the slide- heat fixInsure that shrinkage of the cells does NOT occur- distorts the cells, give improper representation of the cells shape/size-done by air-drying your slide before heat-fixPrepare a thin smear- thick smears make it harder to see individual cells and their arrangement, gives false staining resultsSlide14

Smear Preparation

Solid mediaAdd one drop of water to your slide

Use sterile inoculating needle to pick one colony- just a slight touch neededMix bacteria into water onto slide and try to spread out and thin as possibleLiquid media: no additional liquid is needed

Use a sterile loop, place a couple

loopfuls

of broth onto the slide- sterilize loop between touching the slide and dipping your loop

Place slide on heat plate to dry and heat fix smear (just until slide is completely dry)

Make sure you label your slide so you know which smear is which organismSlide15

Staining

Different types of Staining Simple Stain: use of a single stain to color a bacterial cell

Differential Stain: Use of a combinations of stains to differentiate between 2 or more organismsStructural Stains: stains only one part of a cell so it can be distinguished from the rest of the cell (ex: flagellar)

How do the stains work?

Biological Stains contain chromophores- chemicals that can impart color

A bacterial cell has slight over-all negative charge

Cationic/Basic Dyes: positively charged so it binds to the cell

Anionic/Acidic Dyes: negatively charged; repels the cells, stains everything else, results in a negative/indirect stainSlide16

Simple Stain

Uses only one colorCan only determine size and shapeSlide17

Differential Stain

Uses more than one color with a decolorization step between them

. (will do next lab)Slide18

Today’s Lab

Pure Culture-Split up into groups of 2

TSA (1/Group)Mix 1 (Incubates at Room Temp)

EMB (1/group)  Mix 2 (Incubates at 37º)

Isolation Streak of the assigned mix on each plate

Smear Prep & Simple Staining

- On your own

Pseudomonas aeruginosa

in TSB

Staphylococcus aureus

on TSA

Stains:

Carbolfuchsin

: 1 min

Crystal Violet: 1

min

Safranin

:

15

min

Methylene Blue:

15

min Slide19

Lab

Work on your own. Each student to make:Smear of

Escherichia coli from TSB (tryptic soy broth)Smear of Staphylococcus aureus from TSA (tryptic soy agar)

Each of student in your row will use a different stain

Carbolfuchsin

: 1 min

Crystal Violet: 1 min

Safranin: 15 mins

Methylene Blue: 15 mins

Make notes of your observations; make sure you take a look at your fellow classmates slides to see the different stains

Record your observations on page 43