Lab Practical #1

Question 1

Why special handling of microbes is required in our bio lab

Answer 1

We must be certain to leave no contamination for the others who learn and work in the same room. We must also keep ourselves safe.

Question 2

Safety in the Lab

Answer 2

Hand-washing, goggles, bandages, and gloves are simple measures readily available in the lab. In the event of a spill or splash incident, we have an Eye Wash Station and a show. In the event of a fire, we have a fire blanket.

It all cases, take care of yourself first. If you spill a chemical or splash in your eye, alert teacher as you get yourself to a sink or the Eye Wash Station or shower. If you catch a cuff on fire, stop/drop/roll, and yell for the teacher to get the fire blanket. If you come to lab with a cut, cover it, bandages and gloves are available and no permission is needed.

Question 3

MSDS

Answer 3

Materiel Safety Data Sheets. Standardized summary of physical properties and dangers associated with each chemical.

Found behind the door in the Prep Room. Also on file on each computer.

Question 4

Chemical Hygiene Plan

Answer 4

Instructions for safe handling of chemicals and of emergencies, proper waste disposal.

Found behind the door in the Prep Room.

Question 5

Food

Answer 5

There’s no good, beverage, or gum chewing in lab. This is to minimize the chance for hand to mouth transmission. We use pure cultures of microbes, and so you must be aware of what you’re doing or you can spread a large number of cells with one simple error. Someone inadvertently touching a contaminated surface may introduce microbes if they touch a ;mucous membrane,’ and ‘hand to mouth’ is one direct avenue. We must be careful NOT to dispose of empty food or gum wrappers, or beverage containers in the trash containers that are in Room 309. Use the trash outside the room.

Question 6

4 Likely Points of Introduction of Microbes

Answer 6

Eyes, ears, nose, and mouth.

Question 7

Hypothesis of Experiment 1

Answer 7

Each surface will contain different microbial communities.

Question 8

Null Hypothesis of Experiment 1

Answer 8

The same microbial communities will be found on every surface.

Question 9

Controls of Experiment 1

Answer 9

Air, Cotton Swab, pre-existing colonies in plate, operational (doing all of experiment BUT using a sample).

Question 10

What To Label On Plate Experiment 1

Answer 10

Put your name, date, and where you collected your sample.

Question 11

Animate vs. Inanimate Surfaces

Answer 11

Microbial communities will differ on different surfaces between different surfaces provide different needs that some microbial communities will need and others won’t.

Question 12

Microbes Are Not Just Bacteria

Answer 12

Microbes are microscopic life. As such, a large number are single-celled organisms common in water and soil such as flagellates, ciliates, and amoebae. Most are prokaryotes. A small percentage are simple fungi. A few are animals (infectious as one or a very few cells) and you are most familiar with them as parasites.

Question 13

Hazardous Agents

Answer 13

Biological
- Animals, plants, microorganisms.

Chemical
- Solids, liquids, gases
+ Laboratory safety and chemical waste training.

Radiological
- Isotopes, x-ray or laser equipment.
+ Lab based training with Radiation Safety staff.

Question 14

Physical Hazards

Answer 14

Cuts
- Animal or plant dissections, broken glass, slides.

Fires
- Gas burners, alcohol burners, alcohol containers.

Question 15

Biological Agents

Answer 15

• Microorganisms and their toxins.
• Viruses and sub-viral particles.
• Recombinant products (plant, animal microbial)
• Parasites
• Cultured animal cells and the potentially infectious agents these cells may contain.
• Clinical specimens (tissues, blood, body fluids)
• Tissues from experimental animals
• Allergens

Question 16

Bioharzardous Agents

Answer 16

Biological agents capable of causing disease or death to normally healthy adults.

Question 17

Principles of Biosafety

Answer 17

Know and understand the biology and the infectious or adverse environmental potential of the bilogical agents that you handle.

Always use good microbiological technique (aseptic technique) when handling biohazardous agents or any microorganism.

Use only those disinfectants or sterilants with proven efficacy against the specific biological agent you’re using.

Each worker handling biohazardous material is responsible for their own actions, safety and the safety of others around them.

Supervisors (Lead instructors/Teaching assistants) must properly train their workers/students before permitting them to handle biohazardous agents.

Report all accidents to your supervisor/instructor.

Never permit biohazardous materials to leave the lab without prior decontamination or proper containment.

Never eat, drink, smoke, apply cosmetics, or contact lens in the lab.

Question 18

Disease Transmission Requirements

Answer 18

Presence of infectious agent.

Sufficient infectious dose.

Sufficient virulent capacity.

Access into the body (route of infection)

Host susceptibility to infection.

Question 19

Personal Risk Factors

Answer 19

• Age
• Ethnicity
• Occupation
• Stress
• Poor nutrition
• Pregnancy
• Immunodeficiency
• Alcohol, drug dependency
• Treatment for chronic illnesses

Question 20

Routes of Infection

Answer 20

• Ingestion
• Inhalation
• Parental Inoculation (broken skin)
• Mucous Membrane
• Animal Contact

Question 21

Mode of Transmission

Answer 21

Direct Contact

Indirect Contact
- Person to object to person.

Vectors
- Insects

Question 22

Airborne Transmission

Answer 22

Aerosol

Droplet

Dust

Question 23

Determining the Risk of an Agent

Answer 23

Pathogenicity or the organism.

Mode of transmission and host range.

Availability of effective preventive measures.

Availability of effective treatment.

Question 24

BL1

Answer 24

Suitable for work involving well characterized agents not known to cause disease in healthy adult humans and of minimal potential hazard to laboratory personnel and the environment.

Ex: Bacilus subtilis, Escherichia coli, K-12

Question 25

BL2

Answer 25

Suitable for work involving agents of moderate potential hazard to personnel and the environment. Immunization of antibiotic treatment is available.

Ex: Hepatitis B virus

Question 26

Exposure/Accident Reduction Strategies

Answer 26

Appropriate Engineering Controls

Appropriate Personal Protective Equipment

Good Work Practice Controls Employ Universal/Standard Precautions. When means treating all materials and surfaces as if they were contaminated.

Question 27

Engineering Controls

Answer 27

Equipment and supplies that isolate you from the hazard

Control spills, splashes and aerosols

Disinfect and clean.

Question 28

Personal Protective Equipment

Answer 28

Gloves and Protective eyewear, as needed.

Use the right type and combo for each procedure.

Question 29

Work Practice Control

Answer 29

Appropriate hand washing procedures
- 20 seconds with soap and running water.

Sharps handling
- Use sharps containers.

Appropriate Footwear
- No flip-flops, sandals, slides, or other open toe shoes.

Emergency response procedures (such as first aid or spill cleanup)

Question 30

Reporting Requirements

Answer 30

Spills of cultures or chemicals.

Fires of any size.

Cuts and other injuries.

Illness.

Question 31

Fixation

Answer 31

The process by which the internal and external structures of specimens are preserved and fixed in position. It inactivates enzymes that might disrupt cell morphology and toughens cell structures so that they do not change during staining and observation. A microbe is usually killed and attached firmly to the microscope slide.

Question 32

Heat Fixation

Answer 32

Used to observe bacteria and archaea. Typically, a film of cells is gently heated as a slide is passed through a flame. Heat fixation preserves overall morphology. Although heat fixation inactivates enzymes, it also destroys proteins that may be part of subcellular structures.

Question 33

Gram Stain

Answer 33

Developed in 1884 by Danish physician Christian Gram. It’s the most widely employed staining method in bacteiology. An example of differential staining. Divides most bacteria but not archaea) into two groups, gram negative and gram positive.

1) The smear is stained with the basic dye crystal violet, the primary stain.
2) Followed by treatment with an iodine solution functioning as a mordant. Iodine increases the interaction between the cell and the dye so that the cell is stained more strongly.
3) Smear is next decolorized by washing with ethanol or acetone. This step generates the differential aspect of the gram stain; gram positive bacteria retain the crystal violet, whereas gram-negative bacteria lose the crystal violet and become colorless.
4) Finally, the smear is counterstained with a simple basic dye different in color from crystal violet. Safranin, the most common counterstain, colors gram-negative bacteria pink to red and leaves gram-positive bacteria dark purple.

Question 34

Differential Staining

Answer 34

Procedures used to distinguish organisms based on their staining properties.

Question 35

Streaking

Answer 35

Streaking is a means of dilution, a way to provide genetically pure stock for physiological work, and a means to achieve a pure culture (single species of microbe only). In fact, a colony is a clone.

Question 36

Cleaning Oil Off Objective Lens

Answer 36

Use ethanol (95%) on cheesecloth to start, then ethanol on lens paper, then a huffed lens paper as a blotter to check that all the oil has been removed.

Question 37

Capsule Staining

Answer 37

Simple staining procedure. A technique that reveals the presence of capsules, a network usually made of polysaccharides that surrounds many bacteria and some fungi. Cells are mixed with India ink or nigrosin dye and spread out in a thin film on a slide. After air-drying, the cells appear as lighter bodies int he midst of a blue-black background because ink and dye particles can’t penetrate either the cell or its capsule. Thus capsule staining is a kind of negative staining. The extent of the light region determined by size of capsule and cell itself. There is little distortion of cell shape, and the cell can be counterstained for even greater visibility.

Question 38

Flagella Staining

Answer 38

Provides taxonomically valuable info about the presence and distribution pattern of flagella on prokaryotic cells. Bacterial and archael flagella are fine, threadlike organelles of locomotion that are so slender they can only be seen directly using the electron microscope. To observe bacterial flagella with the light microscope, their thickness is increased by coating them with mordants such as tannic acid and potassium alum, and then staining with paraosaniline (Leifson method) or basic fuchsin (Gray method).