Cab H2 MB

Microbes in the Environment

  • Microbes almost everywhere:

    • Water we drink

    • Air we breath

    • Earth we walk

    • In and on our bodies

  • Microbiome: community of microbes that inhabit a particular environment

    • Environmental microbiomes:

      • Exist on surfaces such as lab benches

    • Human microbes:

      • Exist on human body surfaces

    • Differ from one area to another and from one individual to another individual

    • Ubiquitous microorganism = harmless

  • Lab purpose is to attempt to culture (grow) some microbes from environment

  • When a medium is selected for culturing bacteria, macronutrients (carbon, nitrogen, phosphorus, sulfur) an energy source and any necessary growth factor must be provided

  • Chemically defined medium: medium whose exact chemical composition

Culture Media:

  • Complex media: exact chemical composition varies slightly from batch to batch

    • Most chemoheterotrophic bacteria

    • Protein in the form of meat extracts and partially digested proteins called peptones usually supply carbon, energy and nitrogen sources

  • Nutrient broth: used liquid complex medium

  • Nutrient agar: nutrient broth + agar added → becomes solid medium

  • Agar: from marine red algae

    • Few microbes can degrade agar (remain solid during microbial growth)

    • Liquefies at 100 C and remains in a liquid state until cooled to 40 C

    • Once solidified and can be incubated at temps up to 100 C

  • Media must be sterilized

  • Steam sterilization/Autoclaving : most common of sterilizing culture media that are heat stable

    • Uses steam under pressure

    • Material is placed in the autoclave and heated to 121 C at 15 pound of pressure (psi) for 15 minutes

Growing Bacteria:

  • Petri plates: contain solid media provides large surface area for examination of colonies

  • Microbes will be inoculated or intentionally introduced onto nutrient agar or into nutrient broth

  • Bacteria inoculated Into culture media increase in number during an incubation period

  • After suitable incubation, liquid media become turbid/cloud as a result of bacteria growth

  • Colony: pop of cells that arises from single bacterial cell

  • Colony-forming unity: a colony may arise from a group of the same microbes attached to one another

  • Although many species of bacteria give rise to colonies that appear similar, each colony that appears different is usually a different species

Preparing Culture Media:

  1. Prepare 100 ml of nutrient broth in a 250-ml flask. Using the graduated cylinder, add 100 ml of distilled water to the flask. Read the preparation instruc- tions on the nutrient broth bottle. Calculate the amount of nutrient broth powder needed for 100 ml. If the amount needed for 1000 ml is _____ grams, then ______ grams are needed for 100 ml. Weigh out the required amount and add it to the flask. Stir with a glass rod until the powder is dissolved.

  2. Attach a pipette bulb or propipette to the 5-ml pipette. Pipette 5 ml of the nutrient broth into each of three test tubes, and cap each tube. Label the tubes “Nutrient broth.” Place two in the To Be Autoclaved rack. Label the remaining tube “Not sterilized,” and incubate it at room temperature until the next laboratory period.

  3. Add agar (1.5% w/v) to the remaining 85 ml of nutrient broth.

  4. Bring the broth to a boil, and continue boil- ing carefully until all the agar is dissolved. Be careful: Do not let the solution boil over. Stir often to prevent burning and boiling over.

  5. Stopper the flask, label it “Nutrient agar,” and place the flask with the tubes in the To Be Autoclaved basket.

  6. Listen to the instructor’s demonstration about use of the autoclave.

  7. After autoclaving, allow the flask and tubes to cool to room temperature, or place the flask in a 45°C water bath. What effect does the agar have on the culture medium?______________________ Incubate one tube at room temperature until the next laboratory period.

Pouring Plates:

  1. Obtain a flask of melted nutrient agar from the 45°C water bath. If the agar has solidified, you will have to reheat it to liquefy it

  2. The sterile nutrient agar must be poured into Petri dishes aseptically—that is, without letting microbes into the nutrient medium. Read the following proce- dure before beginning so that you can work quickly and efficiently.

    1. Set four sterile, unopened Petri dishes in front of you with the cover (larger half) on top. Have a lighted laboratory burner or incinerator within reach on your workbench.

    2. Holding the flask at an angle, remove the stopper with the fourth and fifth fingers of your other hand. Heat the mouth of the flask by pass- ing it through the flame three times (FIGURE 3.2a) or passing it over the mouth of the inciner- ator two or three times (FIGURE 4.4b). Why is it necessary to keep the flask at an angle through this procedure?

    3. Remove the cover from the first dish with the hand holding the stopper. Quickly and neatly pour melted nutrient agar into the dish until the bottom is just covered to a depth of ap- proximately 5 mm (FIGURE 3.2b). Keep the flask at an angle, and replace the dish cover; move on to the next dish until all the agar is poured.

    4. When all the agar is poured, gently swirl the agar in each dish to cover any empty spaces; do not allow the agar to touch the dish covers.

    5. To decrease condensation, leave the Petri plate covers slightly ajar for about 15 minutes until the agar solidifies.

    6. Place the empty flask in the discard area.

Inoculate two plates from the environment and two plates from your body. Wet a swab in sterile water and swab the area to be sampled. After swabbing the agar surface with one of the swabs, place the swab in one of the sterile tubes of nutrient broth and leave it there dur- ing incubation. You may need to break off part of the wooden handle to fit the swab into the nutrient broth tube. c. The plates and tube should be incubated at the approximate temperature of the environment sampled. Incubate bacteria from your body at or close to your body temperature. What is human body temperature? ____°C d. Incubate all plates inverted so that water will condense in the lid instead of on the surface of the agar. Why is condensation on the agar un- desirable? _________________________________ e. ________________________________ Incubate all inoculated media at the appro- priate temperature until the next laboratory period.

Second Period:

  1. Observe and describe the resulting growth on the plates. Note each different-appearing colony, and describe the colony pigment (color) and morphol- ogy using the characteristics given in FIGURE 3.3. Determine the approximate number of each type of colony. When many colonies are present, record TNTC (too numerous to count) as the number of colonies.

  2. Describe the appearance of the nutrient broth labeled “Not sterile,” “sterile,” and the broth you inoculated. Are they uniformly cloudy or turbid?________________________________________ Look for clumps of microbial cells, called floccu- lent. Is there a membrane, or pellicle, across the surface of the broth?______________See whether microbial cells have settled on the bottom of the tube, forming a sediment (FIGURE 3.4).

  3. Discard the plates and tubes properly.

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Fungi: Yeast and Molds:

  • Fungi: eukaryotic unicellular/multicellular organism

    • Obtain nutrients by absorbing dissolved organic materials through cell wall + plasma membrane

    • Are aerobic except yeast

    • Kingdom = Fungi

    • Compared to bacteria, fungi grow better in acidic conditions + tolerate higher osmotic pressure + lower moisture + larger (more cellular/morphologic details)

  • Yeast: nonfilamentous, unicellular fungi (spherical/oval)

    • Widely distributed in nature, found on fruits, leaves as white powdery coating

    • Budding yeast → asexually → cell division is uneven

      • New cell form as a protuberance (bud) from parent

      • Some cases bud fails to detach themselves → pseudohypha forms

    • Facultative anaerobes (many industrial fermentation)

  • Molds: multicellular filamentous

    • Thallus: Macroscopic mold colony

      • Composed of mass of stands called mycelium

      • Each strand is a hypha

      • Vegetative hyphae grow in or on the surface fo growth medium

      • Aerial hyphae/reproductive hyphae → originate from vegetative hyphae and produce a variety of. Asexual reproductive spores

      • Hyphal strand of most molds is composed of individual cells separated by a septum

        • Septate hyphae

      • Rhizopus lack septa and are a continuous mass of cytoplasm with multiple nuclei → coenocytic hyphae

    • Identified by:

      • Appearance fo colony (color, size), hyphal organization (septate or coenocytic) and structure + organization of asexual spores

  • Fungi classified into phyla based on similarities in their rRNA eliminated need for deuteromycota (asexual vs sexual)

    • Water molds/oomycota related to algae such as diatoms/kelp than to fungi

      • Form sexual spores → oospores (formed by fusion of two cells motile asexual spores/zoospores)

    • Zygomycota are saprophytic molds that have coenocytic hyphae

    • Saprophyte: obtain nutrients from dead organic matter (in healthy animal and plants, don’t cause diase)

      • Saprophytic molds Usually produce conidiospores asexually (penicillium and aspergillus)

      • Rhizopus/bread mold

    • Asexual spores formed inside a sporangium/spore sac → sporangiospores

    • Sexual spores called zygospores formed by fusion of 2 cells

    • Ascomycota: molds with septate hyphae and some yeast

      • Sac fungi bc their sexual spores (ascospores) produces in ascuas

    • Basidiomycota: fleshy fungi, mushrooms and have sexual spores called basidiospores produced by club shaped stalk called a basidium

  • Use sabouraud agar (selective medium) to culture fungi

    • Simple nutrients (glucose + peptone)

    • Low pH (inhibits growth of many other organism)