Micro Lab test

Aseptic Technique

A way to inoculate something without contaminating the media/sample

Aseptic def

a = without

sepsis = contamination

Use of a Microscope

Grab prepared slide

start at 4x (always start at 4x)

Then increase as needed (10x, 40x, 100x)

First 7 parts of a microscope

1. Occular/eyepiece

2. Body

3. Nosepiece

4. Objectives

5. Stage

6. Stage adjustment knob

7. Light intensity control

next 7 parts

1. Substage condenser

2. Aperture diaphragm control

3. Field diaphragm lever

4. Light source

5. Base

6. Fine focus knob

7. Coarse focus knob

Resolution

The ability of a lens to distinguish separate entities that are very small and close together

Parfocal lens

Lenses that maintain focus as objective lenses are switched (require course and fine adjustments)

Depth of field

depth of field (DOF) is the vertical thickness of the specimen that remains in sharp focus at the same time

Field of view

the portion that is visible

Working distance

the distance between the tip of the objective and the stage

Why would you use oil immersion

to increase image resolution and clarity at high magnification (like 100x) by reducing light refraction

Isolation Steak Plate

How to properly streak a singular organism on an agar plate

• Flame your loop and grab a sample of the organism streak about half the plate

• Flame the loop again, drag the loop across one side of the first streak area cover a quarter of the plate

• Do not flame the loop, drag across the second streak area and streak the final quarter

Results

First streak - heavy growth

Second streak - growth starting to thin out

Third - light growth, individual isolated colonies

Protazoa

Single celled eukaryotic micro-orgs. Live in fresh or saltwater

• many are predators

• Can move via flagella, cilia, undulating membranes, and amoeboid motion

• Some are photosynthetic (dinoflagellates)

Four major groupings of protozoa

• Amoeba proteus - uses amoeboid motion to move

• Paramecium caudatum - moves via cilia

• Euglena - moves with flagella

• Sporozoa - nonmotile

Protozoa experiment and results (ch 5)

• Place prepared slide in microscope

• use 40x and then 100x, up to 400x

• Should be able to distinguish between organism based on movement (or movement accessories)

Major groups of fungi

They are dimorphic - they exist in two forms

• Unicellular: yeasts

• Multicellular: molds

Difference between yeasts and molds macroscopically

Macro

• Yeast:

  • Appearance: Smooth, creamy, and moist.

  • Form: Bacteria-like colonies.

  • Color: Mostly white, off-white, or cream.

• Mold:

  • Appearance: Fuzzy, woolly, or hairy.

  • Form: Filamentous mats that spread.

  • Color: Vibrant colors (green, black, gray) with colorful spores.

Difference between yeasts and molds microscopically

Micro

• Yeast:

  • Cell Structure: Unicellular, round, or oval shapes.

  • Reproduction: Multiplies via budding; may form chains (pseudohyphae).

  • Gram positive

• Mold:

  • Cell Structure: Multicellular, long branching threads called hyphae.

  • Reproduction: Multiplies via asexual spores (conidia/sporangiospores).

  • typically gram negative

What are the vegitative and reproductive structures of molds

Veg - Hyphae and mycelium

Reproductive structures

• Conidiophores: Aerial hyphae that bear uncovered asexual spores called conidia or conidiospores at their tips.

• Sporangiophores: Aerial hyphae that support an enclosed sac called a sporangium, which holds sporangiospores inside.

What is the fungi that produces spores asexually, what are the spores called?

• Fungi: Zygomycetes

• Spores: sporangiophores

Sac fungi that produces asexually

• Ascomycetes

Fungi that reproduces with sexual spores

• Basidiomycetes

Fungi that reproduces with sexual unidentified spores

• Deuteromycetes

What are the features of buds?

• Buds (Blastoconidia):

  • Organism: Yeasts.

  • Shape: Small daughter cell pinching off a larger parent cell.

  • Junction: Narrow, constricted neck at the point of attachment.

What are the features of hyphae?

• Organism: Molds.

• Shape: Long, microscopic, tube-like filaments.

• Junction: True parallel walls with no constrictions at cell borders.

What are the features of Pseudohyphae?

• Organism: Elongated yeast chains (e.g., Candida).

• Shape: Chained segments resembling a string of sausages.

• Junction: Pronounced constrictions at every cell-to-cell junction.

Simple Stain

• stains that use one stain to complete the procedure

• Safranin, or methylene blue (negative charge)

• Only stains the cell not the background

• Useful for observing shape: bacillus, coccus, and spirillum

Simple stain process and results

• Apply a smear onto a slide. Mix culture with a drop of water

• Heat fix the slide after air drying

• Cover smear with either safranin or methylene blue

• Allow it to stain and then rinse with water and dry with bibulous paper

Results

• You should be able to see the shape and arrangement of the cell

Negative Stain

• A staining technique opposite to a simple stain

• Background is colored, bacterial cell is not colored as they repel the acidic chromogen

• Cell shape and size can be recorded

• Good for non gram staining bacteria

Negative Stain procedure and results

• Apply and heat fix bacteria to slide.

• Add drop of nigrosin, pick up a second slide and use to it spread the drop of nigrosin with a 30* angle.

• Allow to air dry

• View with a coverslip and oil immersion

Results

• Form of simple staining just stains the background

• Stain is negatively charged like the bacteria hence it is repelled

• Cells will appear bright white

Capsule Stain Background

• Stain the bacterial cell and background, leave capsule unstained

• Use crystal violet or safranin, rinse with copper sulfate

• Capsules are made of polysaccarides and polypeptides - they are sticky

• Can store both nutrients and water

• Allow cell to adhere to surfaces

• Allow cells to avoid phagocytosis as it makes them difficult to detect

Capsule Stain procedure and expected results

• Add a loopful of inoculum and smear to the size of nickel on slide, should be a thick smear

• Air dry

• Cover with crystal violet or safranin

• Tip slide and rinse with copper sulfate

• Bibulous paper and use oil immersion to view

Results

• Capsule will appear uncolored or light blue

• Useful for identifying type of organism as capsules do not show up on other staining techniques due to heat fixing

Gram Stain Background

• Developed by Has Gram

• First step in identifying most unknowns

• Differential stain that reveals two groups of bacteria: gram positive and gram negative

• Results depend on structure of cell wall

Gram positive

• thick layer of peptidoglycan

Gram Negative

• thin wall of peptidoglycan and has an outermembrane with lipopolysaccharide

Gram Stain dyes

Primary stain: crystal violet is a basic stain and stains all of the cells purple

Mordant: Grams iodine, mordants fix color into the cell wall

Decolorization: acetone-alcohol is used to create large holes in gram-negative cell and washes out grams idonine color

Counterstain: safranin stains the gram negative cell to differentiate

Gram Stain process and results

• mix sample with drop of water on slide and allow to air dry and then heat fix slide

• Cover slide with crystal violet and rinse with distilled water

• Cover with gram’s iodine and rinse with alcohol

• Cover with safranin and rinse with distilled water

• bibulous paper and use oil immersion

Results

• Gram positive will appear purple

• Gram negative will appear pink

Endospore Stain background

• Vegetative cells develop endospores to survive harsh environments

• Coated with keratin and proteins making them durable

• Spores can be located terminally (near the end), Central, or subterminal (between center and end)

• hard to kill with heat, radiation, desiccation, and chemicals

• Can be used as biological weapons (anthrax) botulism is a form of endospores

• Stain is a differential or structual stain

• uses boiling water to penetrate the stain into the spore coat

• Then decolorized and counterstain to stain the veg cells

Endospore Stain procedure and results

• mix sample with drop of water, dry, and heat fix

• place slide over boiling water, place a paper towel over smear, soak with malachite green for 5 minutes over steam

• remove from steam and allow to cool, remove paper towel

• Rinse with distilled water and counterstain with safranin

• Rinse, bibulous paper, oil immersion

Results

• Spores appear blue green

• veg cells appear red

Acid Fast Stain Background

• differentiates between acid-fast (mycobacteria), and non-acid-fast bacteria

• Acid fast cells have a waxy wall, mycolic acid in the cell walls make them resistant to desiccation and hard to stain

• Two methods: Ziehl-Neelsen (uses steaming water), and Kinyoun (uses concentrated reagents - cold method)

• Steam method “melts” waxy layer and traps stain in cell once it cools

• Kinyoun concentrations contain more phenol which is a lipid solvent which allows the stain to stain the cell

Acid fast procedure (Kinyoun)

• mix sample with a drop of water on slide, allow to air dry and then heat fix

• Cover sample with carbol fuchsin

• Rinse with water then decolorize with acid alcohol

• Rinse with water and counterstain with methylene blue

• Rinse, bibulous paper, oil immersion

Results

• Used to view mycobacteria

• Acid fast appear red or fuchsia

• Non-acid-fast appear blue

Blood Agar background

• medium is both enriched and differential

• blood provides extra nutrients needed by fastidious streptococci and other pathogenic bacteria to grow

• Strep and Staph make toxins called hemolysins that break down rbcs causes blood agar to darken around colony (alpha-hemeolysis)

• hemeglobin breakdown causes a green color to appear

• Beta -hemolysins causes complete lysis of rbc and breakdown of hemeglobin leading to colorless colony

• Gamma-hemolysis causes no hemolysis

• Streptococcus pyogenes causes bacterial pharyngitis or strep throat. - shows up as alpha-hemolysis

Blood Agar Procedure

Divide back of plate into quaters

• swab the back of your throat and apply to agar

• Incubate in 37* enviroment for 24-48hrs

Results

• Alpha-hemolysis strept will appear green with a halo

• beta will clear and zone will widen

• gamma will have no change

Mannitol Salt Agar background

• Mannitol Salt Agar is a medium that is both selective and differential

• Medium contains high concentration of salt meaning only halophilic bacteria like staphylococci will grow

• Contains phenol red and minnitol

• mannitol turns pathogenic staph yellow but not non-pathogenic, they stay clear

• phenol red allows for differentiation between pathogenic and non

Mannitol Salt Agar procedure and results

• Mark plate

• aseptically apply sample with loop onto plate with a short streak

• incubate

Results

• pathogenic staph shows up yellow

• non-pathogenic is clear

• Nothing is no reaction/growth

Eosin Methylene Blue Agar Background

• Tests for coliform bacteria (E. Coli) typically fecal coliform

• It is a standard test for water safety

• First presumptive step looks for lactose fermenting organisms if that is positive

• the second confirmatory step looks more specifically for fecal coliform bacteria - we use EMB for that

• Medium is both selective and differential

• inhibits growth of gram-positive bacteria and allows gram-negatives (coliforms)

• medium contains sugar lactose allows for differentiation between non-lactose fermenters, lactose fermenters, and heavy lactose fermenters

• the production of acid while fermenting lactose will trigger pink to metallic green color change

Eosin Methylene Blue Agar Procedure and results

• Mark plate

• apply sample to loop, strike quater

• incubate

Results

• poor growth clear - gram positive inhibited by methylene blue

• gram-negative non-lactose fermenter appears to grow but no color change

• coliform lactose fermenter with pink and purple growth

• E. Coli with green sheen cause by rapid lactose fermentation

MacConkey Agar background

• medium that is selective and differential

• Selective due to the bile salts and the dye of crystal violet and inhibit the growth of gram-positive bacteria but allow gram negatives to grow

• It uses the sugar lactose and pH indicator of neutral red which allows it to differentiate between non-lactose fermenters and lactose fermenters

• Really good for identifying enteric bacteria

• In response to lower pH turns fermenters pink, non-lactose fermenters will be clear or uncolored.

MacConkey Agar Procedure and Results

• Divide plate

• Apply sample with loop in one streak and incubate

Results

• gram positive will grow poorly or not at all

• gram negative that are not coliforms will grow well but colorless

• Coliform bacteria will be pink or red

Brownian movement

Appearance of movement due to water molecules colliding with the cells and make them move.

Hanging Drop Method

• put sample on a coverslip, and secure coverslip to the depression slide with petroleum jelly

• Observe with microscope

Results

• Bacteria will be difficult to notice due to lack of color, but you should be able to determine true motility if a few cells are moving. If many are it’s likely due to brownian movement

• Determines whether or not bacteria is motile

Plate Count Background

• used to estimate the number of microbes in a sample

• only plates with counts of 30-300 colonies are considered to be valid and countable

• to low is TFTC, to many is TNTC

• It is used to estimate in enviromental samples through dilution to make it easier to count, then you multiply the count by the inverted dilution ratio to get your estimate.

Plate Count Procedure

• Label tubes and plates

• using a pipette transfer 1ml of milk to the first sample of 99ml of water

• mix sample into water

• take 1 ml from sample 1 and apply it to sample 2 (9ml of water) mix

• take 1 ml from sample 2 and apply it to sample 3 (9ml of water) mix

• repeat for sample 4

• use a pipette to transfer 0.1 ml to each plate, use glass beads to spread bacteria all over plate

Results

• At lower dilutions you will see plates with over 300 colonies (TNTC)

• Plates with between 30-300 are countable

• Plates with less than 30 are TFTC

Osmotic Pressure and Growth Background

• Osmosis is regulated not by water but by the concentration of solutes contained within that water

• In order to predict movement of water, the salinity of the cell’s cytoplasm must be compared to the salinity or solute of concentration of the environment around the cell

obligate halophile

bacteria that only grow in the presence of salt

Faculative halophile

bacteria that grow in both low and high salt environments

Turgor pressure

created by the influx of water into a bacterial cell

is used to sustain cell life

Plasmolysis

Cell shrinking due to hypertonic reaction to solute concentration outside of cell

Osmotic Pressure and Growth Procedure and Results

• Apply samples of e. coli and s. aureus to a tube of 1%, 3%, 7%, and 11% salt

• Incubate

Results

• measure absorbance with a spectrophotometer

• higher readings indicate higher levels of bacteria

• should be able to measure what levels of salt the bacteria can grow best in

Catalase Background

• hydrogen peroxide is produced as a by-product of metabolic pathways

• H2O2 is considered a reactive oxygen species (ROS) can cause significant damage to species

• Many bacteria produce the enzyme catalase to convert H2O2 to water and O2

• Signs of catalase producing bacteria will be a bubbling (champagne bubbles) after dropping H2O2 on it

• distinguishes between gram-positive cocci like Staph and Micrococcus (catalase positive)

• and catalase negative

Catalase procedures and results

• Apply cells from sample with toothpick onto a slide

• 1-2 drops of H2O2

• watch for bubbling

Results

• Bubbles should appear shortly after applying the H2O2

• Bubble indicate catalase positive, no bubbles indicate catalase negative

Oxidase Background

• In the process of cellular respiration glucose is oxidized. The electrons are then shittled to the electron transport chain

• The final step of this process is to reduce O2

• Cytochrome oxidase is the final enzyme of the electron transport chain that reduces 02 to form Water

• There are different froms of cytochrome oxidase depending on the bacteria

• The purpose of this test is to see if the bacteria has cytochrome c oxidase

• a reagent called oxidase reagent is added to the cells

• if cytochrome c oxidase is present the agent turns purple

• Procedure is used to distinguish among the gram-negative rods

Oxidase Procedure and results

• Add oxidase reagent to a papper towel or filter

• Apply organism to reagent using a loop or toothpick

• reaction should develop in 1 minutes

Results

• if it turns blue-purple it is a positive reaction

• a blue ring developing around the sample is not a positive reaction