Micro Lab 1 practical

What do safety glasses not protect you from

splash hazards

When are thermal and punctured-resistant gloves used

when handeling extremely hot or cold materials or sharps

What to do if expose to flame or chemical happens

yell for help

move to closet shower

remove saturated clothing

drench skin under shower

have someone call 911

how long should you rinse your eyes

at least 10 min

how should you wrap someone that is on fire

put them on the floor and help them roll

what should the chemical hood be open to

8 or 16 in

what to clean work bench with

ethanol or isopropyl alcohol

What works best? Hand washing or Alcohol Based Hand sanitizers?

hand washing

How do we ensure proper hand hygiene?

Hands should be lathered for at least 20 seconds while hand washing

Aseptic Technique

\-Prevents the release of infectious agents from cultures into the environment.

\-Commonly practiced in health care range from sterile methods that exclude all \n microbes to antisepsis (antiseptics). \n -Minimizes the likelihood of contamination

Path of light through the microscope

lamp-condenser-specimen-objective lens-ocular lense

objective lens

Light is refracted through (that means it is bent) and produces a magnified, inverted, real image

ocular lens

shows the virtual image

Resolution

The clarity of the a produced image

Limit of Resolution/Resolving power

the smallest distance two points can be for the \n microscope to resolve them as separate beings. (smaller limit of resolution = better clarity)

tOTAL MAGNIFICATION =

= ocular lens X objective lens

What lens has (Largest Field of View) (Greatest Working Distance)

4x

Parfocal

Microscope stays in focus after scanning objective is in \n focus

Field of view

The area that you can view through the ocular lenses of \n a microscope

Depth of focus

The ability to focus vertically through specimens \n using a microscope.

Working distance

The distance between the objective and the slide

Always focus using the

scanning objective (4x) and the coarse \n adjustment knob

Once in focus, after this point, you should only use

the fine adjustment knob

Coarse adjustment is only for

SCANNING OBJECTIVE

do not’s after focusing a microscope

• Do no move microscope once in focus \n • DO NOT USE THE COARSE ADJUSTMENT AFTER THE SCOPE IS IN \n FOCUS—USE ONLY THE FINE ADJUSTMENT

Bright field Microscopy

what we use in lab - makes the field bright and specimen dark

Dark Field Microscopy

Only light coming from specimen is viewed \n background is viewed as dark. (When background and specimen \n are similar)

Phase Contrast Microscopy

The phase of the light coming into the \n specimen is altered which causes a change in contrast of the image \n perceived as it causes differences in brightness. (we can observe \n subcellular structures of biological specimens without staining and \n heat fixing!

Fluorescence Microscopy

Fluorophores are excited by specifically \n chosen wavelengths of light. The excitation of the fluorophores emits a visible photon of light.

Electron Microscopy

bombards specimen with electrons (high \n magnification)

Scanning Electron Microscopy

observe external features

Transmission Electron Microscopy

observe internal features

Pond Water Lab

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What does pond water contain

multitude of microscopic organism

Pond Water Lab

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Procedure:

We will be looking at a wet mount of pond water and identifying different organisms throughout

\- Prokaryotic (Bacteria/Archaea) or Eukaryotic (Algae, Protozoa, Fungi) \n - Is the organism photosynthetic or not? (Look for green pigmentation) \n - Does the organism/bacterium contain a flagellum for locomotion?

Pond Water Lab

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how to tell if the organism photosynthetic or not?

Look for green pigmentation

Pond Water Lab

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What (Prokaryotic) Domain Bacteria are found in the water

a. Eubacteria (Bacterial cells difficult to identify without stain) \n i. Spheres, Rods, Spirals (Clusters, Chains) Ex: Staphylococcus aureus \n b. Cyanobacteria (Primitive Bacterial cells many times green and \n photosynthetic)

Pond Water Lab

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what (Eukaryotic) Domain Eukarya will be in the water

a. Algae (Kingdom Protista) – can be unicellular or multicellular \n b. Protozoans (Kingdom Protista) – single celled \n c. Fungi (Kingdom Fungi) – multicellular \n d. Invertebrate Animals (Kingdom Animalia) – multicellular

Aseptic Transfer tips

1) Minimize the potential for contamination- Keep your books and other items off of \n your lab bench when working with microbes. \n 2) Be organized- Label your test tubes and plates prior to inoculation. \n 3) Keep test tubes in a rack- these test tubes can leak \n 4) Take your time \n 5) Don’t hold cultures by their cap \n 6) Hold your inoculating loop or needle like a pencil in your dominant hand and \n relax. \n 7) Adjust your bunsen burner to have a nice blue cone

Aseptic Transfer

This is the transfer of microbes from one medium to the next \n without contamination from you, others, or the environment.

Culture

Medium that contains microbes

Pure Culture

Medium containing only a single species

Broths

Grow large numbers of microbes

slants

Grow stock cultures to be stored for weeks

Plated Media usage

Obtaining isolated species, antibiotic testing, and quantifying bacterial densities

when is Aseptic Transfer critical

during inoculation of each type of media

Nutrient broth culture

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inoculate:

Staphylococcus epidermidis

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Broth S. epidermidis to fresh TSA slant using an inoculation loop

Nutrient agar slant culture

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inoculate

Serratia marcescens

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Slant S. marcescens to fresh nutrient broth using an inoculation needle

streak plate theory

You can take a mixed culture and isolate individual bacterial colonies by using a streak plate

Colony

originates from one cell and becomes a mass of cells on the agar plate

Mixed nutrient broth culture example

(E. coli + Chromobacterium violaceum)

control of microbial growth steps

1. Use a sterile swab (Do not Flame it) to transfer your organism to the plate creating a bacterial lawn.
2. Douse your antibiotic discs into your particular germicide should have 3 disks with 3 concentrations of the same germicide)
3. Use forceps aseptically (Flame them) to transfer your disks to the plate)
4. Kirby Bauer, you have a device with your antibiotic disks already. \n a. Zone of Inhibition – clearing around the sidk
5. Incubate and view

diffusion

how molecules cross the plasma membrane

The rate of diffusion is affected by

• Media density \n • Molecule size \n • Concentration gradient

Simple Stains components

1\. Solvent - Water or ethanol \n 2. Chromogen (Contains Chromophore that gives it color) and (Auxochrome which is the charged portion of the chromogen allowing for ionic bonding between chromogen and cell.)

Basic Stains

(Auxochrome is positively charged)(Attracted to the negative surface of most bacterial cells

Examples of stains

Methylene Blue, Crystal Violet, and Safranin

Heat Fixation

his kills the bacteria, makes them adhere to the slide, and coagulates \n cytoplasm proteins to make them more visible. (\*May distort cells) \n

Why Stain:

\-Increase contrast where cells are normally transparent \n - Determine cell size, morphology, and arrangement

basic stains are applied to

to bacterial smears that have been heat fixed.

Goals for simple stain lab

1\. Prepare a bacterial smear \n 2. Perform a simple stain \n 3. Identify the S. epidermidis and S. marcescens and record their morphology and arrangement.

How long is a simple stain

Stain for 60 seconds

Escherichia coli characteristics

Gram - , Bacillus, Can exhibit resistance

Staphylococcus aureus characteristics

Gram +, Cocci, Can exhibit resistance

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Pseudomonas aeruginosa characteristics

Gram -, Bacillus, Can exhibit resistance

Gram Stain Procedure step 1

\-Prepare a Smear \n -Suspend some of the material to be stained in a drop of water on a \n microscope slide, spread the drop to about the size of a dime. \n Allow to air dry. Heat fix by gently warming above a flame

why Gram-positive bacteria stain violet

due to the presence of peptidoglycan in their cell wal

Gram stain Mistakes and Results:

1) Failure to add Iodine: All cells would decolorize and appear gram \n negative \n 2) Failure to add decolorizer – all cells would be violet \n 3) Over-decolorizing – gram positives look like gram negatives \n 4) Under-decolorizing – gram negatives look like gram positives \n 5) Failure to apply safranin – gram positives will be violet, gram negatives would be colorless. \n 6) Reversal of stains – most likely all will be purple because safranin \n would have washed out, even if it didn’t violet color would predominate.

What color do Endospores stain

green

Who and/or what is protected by lab safety protocols?

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* students doing lab activities
* lab instructors and technical assistant
* anyone who walks into the lab
* the physical lab space and the equipment housed within the lab

all

Which of the following represent important safety procedures for microbiology students?

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* Always wear open-toed shoes so organisms do not become trapped in shoes.
* Wash your hands before and after laboratory procedures.
* Disinfect your lab bench at the beginning and end of the lab period.
* Only drink a closed container of water while conducting lab procedures.
* Take extra care around the flame of a Bunsen burner by tying up hair and keeping it from combustibles.

\-Wash your hands before and after laboratory procedures.

\-Disinfect your lab bench at the beginning and end of the lab period.

\-Take extra care around the flame of a Bunsen burner by tying up hair and keeping it from combustibles.

Which of the following are potential hazards which you might encounter in the laboratory?

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* infectious microorganisms
* open flames
* hot surface
* corrosive chemicals
* sharp tools or broken glass
* gamma radiation
* magnetic fields

* infectious microorganisms
* open flames
* hot surface
* corrosive chemicals
* sharp tools or broken glass

In which circumstance is food and/or beverage allowed in the laboratory?

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* never
* if containers are kept out of sight
* if containers are covered
* if containers are sealed

never

A disinfectant is used on your work surface:

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\n - after any spill of live microorganisms.

\-after all work is complete.

\-before the beginning of laboratory procedures.

\-All of the answer choices are correct.

All of the answer choices are correct.

Using a cell phone in lab is permissible since it can be used for timing experiments.

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true/false

false

Which of the following items may be on the laboratory bench top while doing lab work? (Select all that apply)

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* cultures (broth tubes, agar plates, agar slants)
* equipment items specifically needed to complete the lab activity.
* backpack, purse
* pencil or pen
* cell phone

* cultures (broth tubes, agar plates, agar slants)
* equipment items specifically needed to complete the lab
* pencil or pen

What should be done to prepare or deal with a laboratory emergency? (Select all that apply)

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* Know the location of safety equipment such as the fire extinguisher.
* Notify your instructor regarding spills or nonemergency situations.
* Call 911 if an emergency occurs.
* Take antibiotics regularly before attending class in case of spills.
* Conduct all lab procedures under a fume hood.

* Know the location of safety equipment such as the fire extinguisher.
* Notify your instructor regarding spills or nonemergency situations.
* Call 911 if an emergency occurs.

hand washing procedure in the correct order

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Thoroughly wet hands in running water.

Apply soap.

Lather for at least 20 seconds by rubbing hands together, paying attention to the back of hands, nails, and between fingers.

Rinse thoroughly in running water.

Dry hands thoroughly and use a towel to turn off the faucet.

Paper towels should be discarded in a biohazard container after washing hands.

false

Jackie went to the sink at the end of lab to wash her hands. After removing her watch and rings, she turned on the water, wet her hands, added soap and thoroughly rubbed and lathered her hands for 30 seconds, cleaning the backs of her hands, her fingernails, and between her fingers. She then turned off the water, and reached for the paper towels to dry her hands. Identify the correct statement below.

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* Jackie should have used paper towels to turn off the faucet.
* Jackie used the correct protocol for washing her hands.
* Jackie should not have removed her watch and rings prior to washing her hands.
* Jackie should have applied the soap to dry hands, and then added the water for lathering.

Jackie should have used paper towels to turn off the faucet.

Jacob had just finished his lab work and went to the sink to wash his hands. He removed his watch, and added liquid soap to his hands. He then turned on the water and began to rub his hands together, lathering for 20 seconds and cleaning fronts and backs of hands, fingernails, and between his fingers.  He then rinsed his hands in running water, dried his hands with paper towels, and used an additional paper towel to turn off the faucet. Which statement is correct?

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* \n Jacob should have washed his hands for at least a minute.
* Jacob wasted paper towels by using one to turn off the faucet.
* Jacob should have wet his hands in running water before applying the soap.
* Jacob did everything correctly.

Jacob should have wet his hands in running water before applying the soap.

Which of the following should be worn at ALL times in the lab? (Select all that apply)

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* lab coat
* closed-toed shoes
* face mask
* protective goggles

* lab coat
* closed-toed shoes

When should the chemical fume hood be used?

when using hazardous chemicals

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Identify the correct statements about lab coats. (Select all that apply)

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* Lab coats are worn in lab so that students can be correctly identified as biology students.
* Lab coats provide some protection against contamination with infectious microorganisms.
* Lab coats provide some protection against exposure to corrosive chemicals.
* Lab coats are taken home for decontamination.

* Lab coats provide some protection against contamination with infectious microorganisms.
* Lab coats provide some protection against exposure to corrosive chemicals.

Goggles or appropriate safety glasses protect the eyes from (Select all that apply)

* chemical splashes 
* fumes of preservatives used on anatomy specimens.
* UV exposure when using UV radiation in the laboratory
* burns from the hot plate or Bunsen burner

* chemical splashes chemical splashes Correct
* UV exposure when using UV radiation in the laboratory

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Gloves provide protection against (Select all that apply)

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* infectious microorganisms
* contamination of cultures with skin bacteria from the hands
* broken glass and sharps
* exposure to chemicals and stains

* infectious microorganisms
* contamination of cultures with skin bacteria from the hands
* exposure to chemicals and stains

What is used to sterilize infectious items prior to cleaning or disposal?

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* bleach soak
* alcohol soak
* autoclave 
* washing in hot soapy water

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autoclave

Which of these items should be placed in the sharps container?

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* cover slips 
* reusable glass culture tubes
* culture plates

cover slips 

What is the best source of information regarding the potential hazards, proper handling, and disposal instructions for a particular chemical?

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* Wikipedia
* PDR (Physician's Desk Reference)
* SDS (Safety Data Sheet) 

SDS (Safety Data Sheet) 

When is it crucial for you to use protective eye wear in the lab? Select all that apply.

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* when working with corrosive, irritating, or infectious materials that may be a splash hazard
* when using a Bunsen burner
* when using the microscope
* when working with sharp objects

* when working with corrosive, irritating, or infectious materials that may be a splash hazard
* when using a Bunsen burner

All laboratory chemicals can be disposed of by pouring them down the drain.

false

Information on the hazards posed by a chemical used in the laboratory can consistently be found 

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* by asking your instructor.
* on the Safety Data Sheets (SDS).
* posted on the wall of the laboratory.
* in the lab manual.

on the Safety Data Sheets (SDS).

Which of the following items should be disposed of in a biohazard bag? (Select all that apply)

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* used agar plates 
* glass culture tubes
* gloves used while working with infectious microorganisms
* paper towels used to clean up a bacterial spill
* animal tissue
* paper and plastic wraps from sterile swabs, pipettes, and stacks of culture media

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* gloves used while working with infectious microorganisms
* paper towels used to clean up a bacterial spill
* animal tissue
* used agar plates 

What color should the Bunsen burner flame be when sterilizing your inoculation instruments? 

blue

Which objective lens provides the least total magnification?

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* low power
* high power
* scanning
* oil immersion

scanning

When the scanning (4x) objective is used the total magnification will be

40

When the low power (10x) objective is used the total magnification will be 

100x

When the high power (40x) objective is used the total magnification will be 

400x

When the oil immersion (100x) objective is used the total magnification will be 

1000x

The __________ causes the stage to move upward or downward.

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* mechanical stage
* coarse adjustment knob
* iris diaphragm
* objective lens

coarse adjustment knob

Which objective lens provides the highest total magnification?

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* scanning
* high power
* low power
* oil immersion

oil immersion