Tools of the Laboratory Power Point #1: Culturing Microorganisms Microbiology Chapter 3 Tools of the Laboratory Power Point #1: Culturing Microorganisms

Methods of Culturing Microorganisms Microbiologists have to find ways to separate different intertwined microscopic species and grow them in order to study them. Microbiologists use the 5 I’s technique! Inoculation Incubation Isolation Inspection Identification

Inoculation The purposeful contamination of otherwise sterile nutrient media, to allow for growth of microorganisms. Inoculation into a nutrient broth (Liquid). Inoculation onto an agar (nutrient “jello”) plate.

What exactly is Nutrient Media??? A substance that provides all required nutrients for the growth of microorganisms. May be specific or non-specific based on its chemical composition: Specific – Contains nutrients to grow microorganisms of interest (ex. only grows E. coli) Non-specific – Contains nutrients to grow any microorganisms present. The media can be stored in test tubes, flasks or petri dishes and the microorganisms grows within these mediums. (See pictures on next slide.) A gel media thickened with agar and a definite nutrient content is most commonly used in a classroom setting. This will grow pretty much any microorganisms present!

Petri Dishes Nutrient Media Flasks Test Tubes

Incubation After inoculation the microorganism will be let to sit in a heated environment (20oC – 45oC) to replicate and grow to a visible level. The growth will show as cloudiness, sediment, scum or color.

Isolation Sterile Technique must be carefully followed in order to prevent contamination. Specific media types are selected that will allow growth of the microbe of interest and inhibit the growth of other types. Each colony appears as its own circle and contains microorganisms of the same type. Samples from a colony may be taken and studied. Typically the off-white colonies represent neutral or good microorganisms and the colonies that are brightly colored, yellow, red, or green, often indicate a pathogenic microorganism. (Ex. E. coli is yellow) It is very important to know what microorganism you are studying. Example: A medical researcher is attempting to create a medicine to fight a specific strain of staph. If they think they are growing staph and testing the medicine on staph, but contamination unknowingly has occurred, and he is really testing e. coli, the medicine will not end up working to treat staff. Notice how each colony (circle) looks the same. This is because the media selected for one and only one type of microorganism to grow. This is a pure culture.

Isolation continued…. If isolation techniques are done correctly a pure culture (pictured on the last slide) that can be used for study will result. Pure cultures contain one and only one type of microorganism. A mixed culture has two or more specifically identified species of microbe. A contaminated culture may have started pure but unwanted microbes were introduced due to poor laboratory technique. This shows a mixed culture because only two types of microorganisms are growing. The are distinguishable by their color (yellow vs white) and appearance of the colony (smooth vs fuzzy). This shows a contaminated culture because several different microorganisms are growing.

Inspection Slides of the grown microbes are created and viewed to observe the shape and size of the microbes, as well as any unique characteristics.

Identification Biochemical testing is a series of chemical tests run on microorganisms that allow the researchers to determine the species they are dealing with. Biochemical testing is used to make sure that the microbe on the media is indeed what you wanted. Remember, this is VERY important as you want to be sure the microbe you think you are testing truly is what you are testing. The testing is necessary because different species may appear quite similar.

Assignment to Recap Notes & Expand knowledge Assignment to Recap Notes & Expand knowledge! Answer on separate composition paper in complete sentences. 14 FORMAL points What are the five I’s? What is nutrient media? What is the difference between specific and non-specific media? What is incubation and at what temperature does it occur? Describe the 4 patterns of growth in test tubes. You may draw pictures if it helps. What is sterile technique? What is a colony? How do you know if a colony is something harmless or something pathogenic? What is a pure culture? What is a mixed culture? What is a contaminated culture? Why is is so important to be sure you are growing and studying the microorganism you believe you are studying? What is inspection? What is identification?

Tools of the Laboratory Power Point #3: Identification Tests Microbiology Chapter 3 Tools of the Laboratory Power Point #3: Identification Tests

The first step in inspecting a microorganism is looking at it under the microscope. An effective microscope should provide three things: Magnification – enlarging to see detail Resolution - clarity and ability to distinguish between two objects right next to each other Contrast – ability to see different colors / structures in the cells

Slide Preparation The specimen to be viewed is typically prepared by mounting it upon a glass microscope slide Live samples will be prepared in a wet mount so that they can be observed in their natural environment and movement can be observed Slides can be prepared from scratch and fixed to a slide with heat and then stained to give it color (ex. gram staining)

Staining Techniques Simple Stain - A single dye application after heat fixing, makes all cells the same color Differential Stain - uses two different dyes after fixing to provide contrast between cell types or different cell parts within one cell. Gram Stain - Gram staining is the most common technique used for identifying and coloring bacteria. Used to determine if the bacteria are gram positive (antibiotic effective) or gram negative (antibiotic resistant.) Differential Stain Gram Stain

Staining Techniques Continued Acid Fast Stain – reveals bacterial that are resistant to the decolorization step in gram-staining, important because this may show false conclusions in the gram stain test Ex. Was used to identify the bacteria that causes tuberculosis. (Mycobacterium tuberculosis) Flagellar Stain - reveals the presence of a flagellum (singular) or flagella (plural)

Assignment to Recap Notes & Expand knowledge Assignment to Recap Notes & Expand knowledge! Answer on separate composition paper in complete sentences. 15 FORMAL points What is the first step of inspecting a microorganism? What three things should a good microscope provide? Define magnification. Define resolution. Define contrast. Why is it best to place live specimens on a wet mount? How are specimens “fixed” to a slide? Describe a simple stain. Describe a differential stain. Describe a gram stain. What is the difference in the structure of a gram positive vs gram negative bacterial cell? At the end of gram staining, what color do gram positive bacteria appear? What about gram negative? How is gram staining used to determine whether or not an antibiotic will be effective in treating a bacterial infection? Describe an acid fast stain. Describe a flagellar stain.