Pure Culture Techniques **Turn on Incinerators and hot plates (low heat) ** Pure Culture Pure Culture Techniques

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

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 colonies Broth: liquid media (same as agar without the thickening agent)

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 chemicals Differential: 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) organisms

Types of Agar Tryptic Soy Agar (TSA) : Supportive media; general media used to grow most microorgansims

Types of Agar Eosin Methylene Blue (EMB): Selective and Differential Selects 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 → colorless

Pure Cultures Needed to identify bacteria and for antibiotic sensitivity Can be achieved by: Pour plates Isolation Streaks

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.

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.

Making a Streak Plate

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

Smear Preparation and Simple Staining Exercise 6

Smear Preparation The way a smear is prepared will determine how well your stain will come out 3 goals to a good smear Making sure the cells adhere to the slide- heat fix Insure 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-fix Prepare a thin smear- thick smears make it harder to see individual cells and their arrangement, gives false staining results

Smear Preparation Solid media Add one drop of water to your slide Use sterile inoculating needle to pick one colony- just a slight touch needed Mix bacteria into water onto slide and try to spread out and thin as possible Liquid 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 organism

Staining Different types of Staining How do the stains work? 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 organisms Structural 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 stain

Simple Stain Uses only one color Can only determine size and shape

Differential Stain Uses more than one color with a decolorization step between them. (will do next lab)

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

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