Unit 1 Lab

Wet Mount, Hanging Drop, Flagella Stain, Culture Method

What are the 4 basic methods to determine motility?

Wet Mount

A drop of the culture on a slide, covered by a cover slip

Hanging Drop

Uses a depression slide to observe bacteria in a drop of water

Flagella stain

Stains the bacteria to look for the presence of a flagella

Culture Method

Uses a motility media (semi-solid agar) to test for the ability of the bacteria to move beyond the inoculation stab line

True Motility

Indicates presence of locomotor organelle (flagellum)

• Self-propulsion towards nutrients or way from toxic substances

• Truly motile cells flit,dart,tumble, or woblle around.

Over-inoculation

Bacteria overcrowded and can’t move which creates a false negative

Old Culture

Bacteria sick/dying and can’t move which creates a false negative

Cells stick to glass slide

Cells can’t move which creates false negative

Receding water line

Bacteria herded by water and appear to move which creates a false positive

Brownian motion

Collision of water molecules makes organisms appear to move which creates a false positive

Ubiquitous

Present Everywhere (Hydrothermal vents, Salt flats, Hot springs, Sulfur Acid Pools, Antarctica)

Saprophytes

Microbes in nature which breakdown decaying organic material into usable material. These help to recycle nutrients

Commensals

Microbes that live on or in an organism that cause no harm, but provide no benefit either

Mutualists

Microbes that live on or in an organism where both the microbe and the host benefit

Obligate Pathogens/Parasites

Organisms that cause disease due to their need to persist inside a host

Opportunistic Pathogens

Microbes that can cause disease under proper conditions. This group can include saprophytes, commensals, and mutualists

• Proper portal of entry

• Acquiring virulence factors

• Infectious dose

• Immunosuppression

Four opportunity factors for opportunistic pathogens

Culture

Group of microbes living together

Culture medium

Nutrient material for the growth of microbes

3 forms of Media

Solid, liquid, and semi-solid

Agar

A carbohydrate (hydrocolloid gel) derived from an algae that is indigestible for most bacteria, but it can still melt. (Melts at 100 C and Solidifies at 42 C

Microbial nutrient Requirements

Energy source, Carbon (found in all organic compounds), Nitrogen (found in proteins and nucleic acids), Sulfur (found in some proteins), oxygen, temperature, pH, etc

Fomite

A non-biologic object that might have microorganisms on it

Colony

Billions of cells that originate from one parent cell

What to include when describing a colony?

Date, time, temp, genus and species (if known)

Lab experiments, Medical procedures, Manufacturing of food

When to use aseptic technique

Aseptic Techniques

Work area disinfection, Sterilizing loops before inoculation, culture tube lip flaming before and after inoculation, covering Petri plates during inoculation, working under the Bunsen burner

Sterilization

Destruction/removal of all forms of microbes (expect prions in most cases)

Heat, filtration, radiation

Examples of sterilization

Boiling

10-30 minutes of this kills most microbes but not those in endospore form or infectious proteins prions

Autoclaving

High pressure steam (heat) sterilization kills everything but some prions

Pure Culture

When all cells of a colony or culture come are descendants of a single or attached organism

Macroscopic Purity

Colony morphology or cultural characteristics are species specific.

Microscopic Purity

Cell morphology can be easily identified and is unaffected by
other organisms

Physiological Purity

Physiological tests are true to the species being studied

Genetic Purity

The sample can be used to evaluate organism’s genome

Streak Plate

Quadrant stream method of obtaining isolated, single colonies from a possible mixed culture/sample

Bright field Microscope

1. Requires staining

2. Requires heat fixation to secure object on slide during oil immersion

3. Staining and heat fixing can damage cell morphology and can kill the cells

Compound Light Microscope

1. Light is passed through the slide

2. Light is not scattered by the object and enters the lens

3. Objective lends

4. Ocular lens

Objective lens

Produces real image of object inside microscope (Objective magnification)

Ocular lens

Produces virtual image seen by observer. (Objective x ocular magnification)

Dark Field Microscopy

1. Light is passed through the slide from a side angle

2. Light scattered by object enters the lens

3. Light not scatted by the object is not captured

4. Advantage: Can be used for observing live and unstained objects

5. Disadvantage: Attachments increase microscope cost

Dark Field Microscope

Advantage: Can be used for observing live and unstained objects

Phase-Contrast Microscope

1. Uses alternate phasing of light wavelengths to hit object at different angles

2. Used for overserving live and unstained objects

Phase-Contrast Microscope

Advantage: Phase shift of light show subtle differences in internal density, showing more features and details

Fluorescence Microscope

1. Uses photoluminescence to observe objects

2. Monochromatic (single wavelength) light is used to excite fluorochromes on object

3. The sample is often treated with antibodies with fluorochromes that bind to cell components

4. The monochromatic light excites the fluorochromes, causing them to emit photons of light of a different wavelength

Fluorescence Microscope

Advantage: Specific features of the object can be identifies and light does not have to pass through specimen

Electron Microscope

1. Electrons are fires at the sample from a cathode

2. Image is magnified through a magnetic coil and captured on a photographic plate

3. Transmission or scanning techniques can show interior or surface, respectively

Electron Microscope

Advantage: High magnification (10^6 x) and resolution

Electron Microscope

Disadvantage: Cost and time, Sample is destroyed in process

Interpupillary adjustment

Adjust distance between ocular lenses depending on distance between your eyes

Diopter adjustment ring

Allows left eye to be focused independently of right eye

Ocular lens

Eye piece lens; magnifies 10x

Objective lens of 4x

Scanning

Objective lens of 10x

Low Power

Objective lens of 40x

High Power

Objective lens of 100x

Oil Immersion

Total Magnification

Ocular magnification * Objective Magnification

Stage

Place for slide/specimen to lay

Slide Holder/Stage Clip

Holds slide and specimen for the mechanical stage

Lamp or Illuminator

Illuminates specimen

Base

Supports microscope and used to carry microscope

Stage Adjustment (X-Y Axis)

Moves stage right or left and backwards or forwards

Iris Diaphragm

Regulates amount of light that reaches specimen

Magnification

Increasing the virtual size of the specimen

Resolution

The ability of a lens to distinguish between 2 adjacent points

Resolving Power (R.P)

The minimum distance 2 points can be apart, and the lens still has resolution

Contrast

Difference between lights and dark (Low contrast=dark)

Refraction

Bending of light as it moves from one medium to an other

Numerical aperture (N.A)

Light capturing ability of lens

Working distance

Distance between objective lens and specimen

Depth of field

Thickness of specimen that is in focus at one time (At higher magnification you can only focus on one thread below)

Range of field

The area of the slide shown in the observed image

Increases

Working distance, depth of field, and range of field all decrease as magnification ____

Parfocal

A microscope that stays in focus when the magnification (lens) change

Unicellular or multicellular eukaryotic

Algae (unicellular/multicellular/prokaryotic/eukaryotic)

Cellulose

Algae cell wall material

Photoautotrophs

Algae feeding

Flagella

How do algae move?

sexual and asexual

Algae reproduction

• Base of the food chain (phytoplankton)

• Produces 50% of atmospheric O2

• Cyanobacteria aid in nitrogen fixation cycle

Positive impacts of algae and cyanobacteria

• High densities, some can produce dangerous toxins (ex. dinoflagellate red tides)

• Can remove oxygen from water when rapidly growing, killing off other organisms

Negative impacts of algae and cyanobacteria

• Food

• Abrasives

• Cosmetics

• Solidifiers

• Nutritional supplements

• Fertilizer

• Biofuel production

Industrial/Commercial Roles of algae and cyanobacteria

Cyanobacteria

The only photoautotrophic prokaryote capable of producing oxygen

Cyanobacteria

Anabaena is a type of what?

Unicellular eukaryotes

Protozoa (unicellular/multicellular/prokaryotic/eukaryotic)

Protozoa

Motile, animal-like, using pseudopods, flagella, or cilia to move

Protozoa

Heterotrophic Saphrocytes (absorbing or ingesting nutrients)

Protozoa

Trophozoite (Feeding/growing stage, active infection in pathogenic)

Feeding/growing stage, active infection in pathogenic

Trophozoite

Cyst

Dormant resistant form, pathogen transmission

Protozoa

Cysts are the dormant form for which microbe

Sexual and asexual

Protozoa (reproduction)

Schizogony

A multiple fission process where the nuclei replicate several times before cell division

Fission, budding, schizogony

Three ways that protozoa reproduce asexually

Unicellular or multicellular eukaryotes

Fungi (unicellular or multicellular/ prokaryotic or eukaryotic)

Chitin, mannas, glucans

Fungi cell wall composition

Hyphae (multicellular) and yeast (unicellular)

Forms of fungi

Sexual and asexual

Fungi (Reproduction)