Micro Lab Final

Neg stain B.subtillis

Neg stain R. Rubrum

Neg Slain S. agalactiae

Neg. Stain S. cerevisiae

Neg stain e.coli

Gram-neg spirillum

Gram-neg coccus

Gram-neg bacillus

Gram pos coccus

Gram pos bacillus

Bacteria gram stain

Bact. Spores

Bact. Capsules

Pertrchicous bacteria

Peritrichous Bacteria

Saccharomyces cerevisiae

Spirillum

Paramecium

Mueller-Hinton agar plates

specialized, non-selective, and non-differential microbiological culture media designed for antimicrobial susceptibility testing (AST), specifically the Kirby-Bauer disk diffusion method

Blood agar plate

enriched, differential culture media used to grow fastidious microorganisms and detect hemolytic activity

Molten agar plates

Poured in a sterile environment (near a flame or laminar flow hood) at 20-30mL per plate and allowed to soldofy for 30-60 minutes.

EMB agar plates

a selective and differential medium used to isolate fecal coliforms and gram-negative enteric bacilli, particularly E. coli.

MacConkey agar plate

isolation of gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non- fermenting gram-negative bacteria

Mannitol salt agar plate

a selective and differential medium used to isolate Staphylococcus species, particularly S. aureus. It contains 7.5% NaCl, which inhibits most bacteria. Mannitol fermentation produces acid, turning the phenol red indicator yellow, indicating S. aureus. Non-fermenters, like S. epidermidis, grow as small pink colonies

Lawn Inoculation or spread plate

A metal triangle is used to spread bacteria on a plate.

Disinfect the forceps

1) The cell spreaders and forceps will be found at one end of the bench.

2) The container of alcohol for you to dip the cell speaders and forceps in will stay at that end of the bench. Keep the container covered and keep the alcohol lamps at least half a metre away from it.

3) When you need a cell spreader or forceps dip it in the alcohol container and carry it to your place at the bench to burn the alcohol off using your alcohol lamp.

Types of parts (disk-diffusion method)

• Zone of inhibition

• Bacterial growth

• Antibiotic disk

Range (Standard plate count)

25-250 range of bacterial colonies.

EMB agar

• Selective and differential medium.

  • selective for gram-negative bacteria

• The methylene blue inhibits the growth of gram-positive bacteria.

• Eosin (dye that responds to changes in pH, going from colourless to black under acidic conditions.

• Contains lactose and sucrose, which are fermentable and encourage growth of some gram-negative bacteria, especially fecal and nonfecal coliforms.

Water tests (results)

• Shows lactose-fermenting gram-negative bacteria acidify the medium and produce a dark purple complex (usually associated with a green metallic sheen on the surface).

Metallic green sheet indicates what

Vigorous lactose and/or sucrose fermentation, typical of fecal coliforms.

Water Test (results)

• A lower level of acid production, due to slow lactose-fermenting organisms, results in a brown-pink coloration of the growth.

• Colonies of non-lactose fermenters appear translucent or pink.

Tyndallization

Requires exposure of the material to free-floating steam for 20 minutes on 3 consecutive days with intermittent incubation. Steaming kills all vegetative cells. Any spores that may be present germinate during incubation and are destroyed during subsequent exposure to a temperature. Repeating this procedure for 3 days ensures germination of all spores and their destruction in the vegetative form.

Autoclaving

Free-flowing steam under pressure requires an autoclave, a double-walled metal vessel that allows the steam to be pressurized in the outer jacket. At a designated pressure, saturated steam is released into the inner chamber, which has been evacuated of all air. The steam under pressure in the other vacuumed inner chamber is now capable of exceeding the boiling point.

Pasteurization

Exposes fairly thermolabile products such as milk, wine and beer for a given period of time at lower temperatures, but high enough to destroy pathogens and some spoilage-causing microorganisms that may be present, without necessarily destroying all vegetative cells. There are two types of pasteurization: high temp., short-time (flash) procedure requires a temp for seconds, and the low-temp, long-time method.

Before you put the microscope away

1) Clean 100X lens

2) Center stage

3) Lower the stage away from the objectives

4) Rotate the low-power objective into place

5) Reduce light intensity

6) Turn off the light switch

7) Cover the microscope

Acetic (Broth, slant, and deeps)

• Broth: turbid with sediment

• Slant: Spreading with discoloration of the media

• Deep: obligate aerobes (acting like facultative anaerobe)

B. Subtilis (Broth, slant, and deeps)

• Broth: Pellicle with sediment if the pellicle is large

• Slant: Filiform with waxy surface, somewhat echinulate

• Deeps: Obligate aerobe

E.Coli (Broth, slant, and deeps)

• Broth: Sediment

• Slant: Filiform with some spreading

• Deeps: Facultative anaerobe

K. rhizophila (Broth, slant, and deeps)

• Broth: Pillicle with sediment if the pellicle is large

• Slant: Filiform with surface beads over time

• Deeps: Obligate aerobe (microaerophilic)

A.aceti (Diameter, form, elevation, margin, and color)

• Diameter: Big/spreading

• Form: Cir/irregular

• Elevation: Raised

• Margin: Undulate

• Colour: Dull/whitish

B. subilis (Diameter, form, elevation, margin, and color)

• Diameter: Bigger

• Form: Circ/irregular

• Elevation: Flat and waxy

• Margin: undulate/lobate

• Colour: Waxy/whiteish

E.coli (Diameter, form, elevation, margin, and color)

• Diameter: Bigger

• Form: circular/irregular

• Elevation: Raised

• Margin: Entire/undulate

• Colour: Shiny/whiteish

K. rhizophila (Diameter, form, elevation, margin, and color)

• Diameter: Pinhead

• Form: circular

• Elevation: convex

• Margin: entire

• Colour: dull/yellow

Using a microscope (steps)

1) Start with the lowest power objective lens (4x)

2) Move the microscope slide around until the sample is in the centre of the field of view

3) Move the focus knob until the image comes into focus

4) Adjust the condenser and light intensity for the greatest amount of light and the clearest image.

5) Change to the newt higher power objective lens. Readjust the focus and/or the condenser and light intensity.

6) Repeat the above with each higher power objective lens in place until you reach the 100X oil immersion objective lens.

Oil Immersion lenses (steps)

1) Swing out the 100X objective halfway

2) Use a single drop of oil

3) Use only a fine focus

4) You do not need a cover slip unless you are saving the slide

6) If you accidentally drag the 40X lens through the oil, clean right away

7) Never let the objective lenses touch the slide.

8) Clean the 100X objective when you are done at the end of the lab.

9) When finished, lower the stage, click the low-power objective lens into position and remove the slide.

Heat fixing

using the alcohol lamps or incinerators

Differetial staining

A differential stain, such as the Gram stain, directly stains the bacteria. It also stains some bacteria one colour and other bacteria a different colour, depending on their characteristics.

Crystal violet (gram stain)

• A basic stain

• Purple

Iodine (Gram stain)

• Mordant

Ethanol (Gram stain)(disinfectant)

• Destaining

• Differential

  • Gram + = purple

  • Gram - = clear

Safrinin (Gram Stain)

• Basic counterstain

• Result:

  • Gram + = purple

  • Gram - = pink

Gram stain

Spore Stain

Differential staining (capsule stain)

Differential Staining (Acid Fast stain)

Shapes of bacteria

• Spirillum

• Bacillus

• Rod

• Coccus

Simple Staining (used for)

It is an approach to staining bacteria that is used to stain bacteria (direct stain) and is used to determine morphology, size and arrangement of the organisms.

Simple staining (steps)

1) Place in methyl blue

2) Rinse off with distilled water until clean.

3) Blot the slide with bibulous paper

4) Use a microscope

Gram viaible

Does not react with Gram staining. This includes:

• Archaebacteria

• Mycoplasmas

• Gram-variable bacteria.

Differential staining (Gram stains)

Are a type of test to determine whether a bacterium is gram-negative (pink/red) or gram-positive (purple). If it does not react, it is gram-variable.

Gram negative is what in a gram-stain

It is pink/purple

Gram positive is what in a gram-stain

It is purple

Differential staining (Gram stains)(steps)

1) Place the slide in crystal violet

2) Rinse with distilled water

3) Mordant (place in grams iodine)

4) Rinse with distilled water

5) Drip decolorize down slide until clear

6) Rinse with distilled water

7) Counterstain (place in safranin)

8) Rinse with distilled water

9) Dry with bibulous paper

10) Use a microscope

Differential staining (Gram stains)(simple steps)

1) Crystal violet

2) Mordant (iodine)

3) Decolorize

4) Counterstain (safranin)

Spore Stain

This type of stain uses heat to aid penetration of the spore wall by malachite green and safranin, staining the bacteria. The specimen in this stain is bacterial endospores, which live as a means of survival of the bacteria and are therefore highly durable.

Capsula Stain

This stain is an important factor in identifying whether a bacterium is pathogenic or non-pathogenic.

Acid Fact Stain

Used to show characteristics of acid-fastness. In such tests, done to rule out tuberculosis.

Be Sure to Know Where These Are:

• The location of the safety shower

• The eye wash station

• The fire extinguisher

• The first aid kit

When Entering the Lab

1) When you first enter the lab, place your backpack, coat and anything else you are carrying under the lab bench.

2) Choose a lab cost from the costs hanging on either side of the microscope shelves.

3) Then wash your hands using antibacterial soap.

4) Go to your place and wipe down the bench using PREempt.

When Exciting the Lab

1) When you have completed your work, wipe down the bench again with PREempt.

2) Hang up your lab coat

3) Pick up your stuff and leave

Purpose of Aseptic Technique

• Protect your work from contamination

• Protect yourself from the bacteria you are working with

• Protect the people you come in contact with from the bacteria you are working with.

Lab Containment Level 1 Minimun Requirements

A. Must wear a lab coat in the lab at all times.

B. Must wear safety glasses when working with liquid cultures.

C. Cannot eat or drink in the lab.

D. Any open cuts or scrapes must be covered with a waterproof dressing.

E. Only necessary items on the lab bench.

F. Long hair must be tied back.

G. No open-toe shoes.

H. No contact lenses.

I. No elaborate rings or dangly jewellery.

Cell Phone is the lab

• Need to be disinfected when entering the lab and disinfected again just prior to leaving the lab

• Put 70% ethanol on a paper towel or kimwipe and wipe down the phone

• While in the lab, the cell phone must be kept in a plastic bag in your lab coat pocket.

Four specified disposal sites for waste

• Two types of sharps containers

  • Only glass sharps

  • Only metal sharps

• Solid waste buckets

• Plate waste buckets

• Tube waste

Two types of sharps containers

All sharp objects, including microscope slides and other glassware, are to be disposed of in the sharps containers provided on each bench.

• Only glass sharps

• Only metal sharps

Only glass sharps

Go in the glass sharps container

Only metal sharps

Go in the metal sharps container.

Solid Waste Buckets

• All contaminated swabs, contaminated pipette wrappers and any paper towels used to clean up bacterial spills

• NOT paper towels used to dry hands or to disinfect the bench, instead, put these paper towels in the regular orange garbage bin.

Plate Waste Buckets

All petri plates are to be disposed of in the buckets labelled “PLATE WASTE”.

Tube Waste

• All tubes are to be placed in the racks on the side bench underneath the sign labelled “TUBE WASTE”. The tubes should be sorted according to size.

To properly mark it must include:

• Name and bench, if applicable

• Date it was inoculated

• Name of the bacteria or exercise

Once finished with observations

• Dispose of all media in the proper waste disposal sites

If you spill the bacterial culture

1) Tell Instructure, and it will help you.

2) If there is no one, follow the lab manual. Use first ethanol and then PREempt.

Oxygen Requirements (organisms)

• Obligate aerobes

• Obligate anaerobes

• Facultative anaerobes

• Aerotolerant anaerobes

• Microaerophiles

Obligate Aerobes

• This includes the organisms that need oxygen to grow/survive.

• See all the material toward the top of the media.

Obligate Anaerobes

• This includes the organisms that do NOT need oxygen to grow.

• Appear only at the bottom of the media.

Facultative Anaerobes

• Organisms can grow in the presence or absence of oxygen.

• In this situation, a bacterial culture will appear most abundant at the top of the media, then gradually decrease as it moves downward.

Aerotolerant Anaerobes

• They can live in the absence of oxygen (they can survive in its presence but do NOT use it).

• In a tube, a bacterium will have a uniform abundance throughout the media.

Parafilm

It is used to seal the plates if the bacteria on the plate are unknown.

Potential Results (Both characteristisc)

• Pellicle

• Flocculant

• Turbid

• Sediment

Pellicle (Broth Characteristics)

• Thick growth at the top of the media

Flocculant (Broth characteristics)

• Flaky aggregates disperaed throughout tube

Turbid (Broth characteristics)

• Growth throughout the tube

Sediment (Broth characteristics)

• Growth at the bottom of the tube.

Potential Results for Bacterial Growth on Slants

• Filiform

• Arborescent

• Beaded

• Effuse

• Rhizoid

• Echinulate

Filiform (Growth on slants)

• Following the line of the original streak.

Arborescent (Growth on slants)

• Treelike growth

Beaded (Growth on slants)

• Nonconfluent to semiconfluent colonies.

Effuse/Spreading (Growth on slant)

• Thin spreading growth