Labs 1-3,6,7,10-15

Question 1

What are microbes?
The five major types?

Answer 1

Microbes are tiny living things that are found all around us and are too small to be seen by the naked eye. all these microbes can be grouped into five major types: Viruses, Bacteria, Archaea, Fungi, and Protists.

Question 2

What are some ways that microorganisms are important to the environment?

Answer 2

1. Cause disease in plants and animals
2. Be used to produce antibiotics
3. Used to produce food products
4. Used in industry
5. Important in nutrient cycling

Question 3

Why are microorganisms the most successful life forms on Earth?

Answer 3

Different microbes can survive and grow under conditions of extreme pressure, temperature, and pH. The can utilize a variety of chemical compounds as nutrients. They are isolated from all environments such as soil, water, air, and plant and animal hosts.

Question 4

Examples of microorganisms? Which are Eukayrotes and Prokaryotes?

Answer 4

Eukaryotes (fungi and protozoans), Prokaryotes (bacteria) and archaea

Question 5

What does ubiquitous mean and why do you think bacteria are ubiquitous?

Answer 5

Ubiquitous mean present everywhere. They are ubiquitous because they have adapted to live in different environments

Question 6

How does the type of media influence the species isolated?

Answer 6

The type of media determines what microbes will grow.

For example, PDA favors fungal growth and TSA favors bacteria growth
BHI: wide variety of organism types including bacteria, yeasts, and fungi

Question 7

What do these abbreviations (PDA, BHI, and TSA) stand for?

Answer 7

PDA- Potato Dextrose Agar
BHI- Brain-Heart Infusion
TSA-Tryptic Soy Agar

Question 8

Normal Microbial Flora/ microbiome/microbiota

Answer 8

Bacteria inhabit every surface of your body at all times. Organisms are a permanent part of your normal microbial flora, and they are harmless or even beneficial for you;

Question 9

Which plates are incubated upside down and right side up and why?

Answer 9

PDA: right side up
TSA: upside down
BHI: upside down
TSA & BHI- upside down- prevent contamination and dryness
PDA- right side up prevents spread of spores produced by fungi
Because of the way the organisms grow. The plates are incubated right side up because the fungi on the plates produce spores and keeping the plates right side up prevents their spread. The plates are kept upside down to conserve the moisture on the plate.

Question 10

Why did we change the incubation temperature for different activities

Answer 10

Different microbes grow at different temperatures.

Question 11

Why do we use a microscope?

Answer 11

to accurately observe and identify microbes/To see things that we cannot see with the naked eye.

Question 12

What are the two types of microscopes you used and how do you compare/contrast?

Answer 12

Dissecting microscope: to view larger specimens
magnify between 10 and 200x more than naked eye and has no objectives
has a lower magnification ability

Compound microscopes: to view much smaller specimens
magnifies 100-1000x with multiple objectives

Question 13

How do you calculate the total magnification?

Answer 13

-ocular lens x objective lens
-Ocular lens is always 10 x

Question 14

Mention the top 3 important parts of the microscope.

Answer 14

Objective – the lens closest to the slide
* Magnification – the size of the specimen under
the microscope
* Total magnification – the product of the objective lens and the ocular lens. If the ocular lens is 10x and the objective lens is 4x then the total magnification is 40x.

Question 15

Define the following terms: objective, nose piece , ocular, stage, stage clip, coarse and fine adjustment

Answer 15

objective: the lens closest to the slide with the specimen on it
nose piece- the platform that can be rotated to switch from one objective to another
occur aka eyepiece: magnify the specimen at 10x
Stage: What the slide sits on
Stage clip :What holds the slide in place
Coarse and fine adjustment :How you adjust the focus of the viewing area

Question 16

the three objectives on a compound microscope

Answer 16

10x (low power obj), 40x (high dry obj),100x (oil immersion)

Question 17

the three objectives are parfocal meaning?

Answer 17

means that once the low power obj has been focused sharply on the specimen, the high dry and oil immersion objs will also be approximately in focus too, should only need to use fine adj knob

maintaining focus even if magnification has changed

Question 18

shifting to an obj with higher magnification decreases the size of?

Answer 18

the field of view, position specimen in the center of the field of view before switching objs

Filed view-the part of the slide that is being viewed through the objectives

Question 19

when high power objs are used less light enters the optical path. The amount of light entering the microscope can be adjusted by

Answer 19

opening the condenser diaphragm/condenser adjustment

Question 20

How to increase the quality of an image

Answer 20

by altering the magnification, resolution (distance between 2 objects), and sharpening the contrast (light difference between two objects)

Question 21

What is the difference in size among blood cells, yeast, and bacteria

Answer 21

viruses are the smallest bacteria are second smallest, eukaryotes yeast and rbcs
yeast are smaller than rbcs

Question 22

What is a wet mount ?

Answer 22

living specimens prepared in an aqueous solution and viewed under a microscope. Typically used to observe motility of an organism.
A wet-mount slide is when the sample is placed on the slide with a drop of water and covered with a coverslip, which holds it in place through surface tension.

Disadvantage: Hard to view due to increased movement of the specimen.

Question 23

What is the difference between a wet mount and smear prep?

Answer 23

During smear prep when bacteria is smeared across a slide and affixed to a slide for observation, it must be fixed and this causes distortion. whereas in wet mouth there is no fixing.

wet mouth allows you to see the cellular arrangements/ true sizes and shapes without distorting

Question 24

What happens when a microbe dries out? How does this happen?

Answer 24

most microbes will die
due to the heat from the microscope light

Bright lights can dry out wet mounts and heat up microorganisms

Question 25

Are wet mounts heat-fixed? Are wet mounts stained?

Answer 25

NO! Heat-fixing the slide would kill the microorganisms on the slide, and defeat the purpose of using a wet mount. NO! Staining techniques would kill the microorganisms on the slide, and defeat the purpose of using a wet mount.

Question 26

Turbid meaning?

Answer 26

Turbid- the cloudiness of a solution. Can indicate the presence of numerous microbes, sediment, or other contaminants.

Question 27

Why do we use aseptic techniques?

Answer 27

to prevent contamination of media, bacterial cultures, and sterile equipment by unwanted microbes

Question 28

how do we use aseptic techniques?

Answer 28

inoculation loop To inoculate or transfer microorganisms from a culture to a sterile growth medium. need incinerator Flaming in between each use

Question 29

how to know when aseptic technique is done right?

Answer 29

When left with a pure culture in which only one bacterial species is growing and it turning red on TSA slant and TSB broth

Question 30

Define these terms: Aseptic, Sterilization, and disinfectant

Answer 30

Aseptic- free from contamination and harmful microbes. Does not mean completely free of all microbes ◦ Sterilization - free from ALL microbes, both harmful and beneficial ◦ Disinfectant- removes some microbes, but not all sterile environment is completely free of microorganisms, aseptic describes something that has been made contamination-free

Question 31

What is streak plating?

Answer 31

The isolation of individual colonies from a mixed culture by progressive dilution on a petri plate(TSA)

Question 32

Why is it important to flame your inoculation loop between the streaking of each sector?

Answer 32

sterilizing

Question 33

how do you label a Petri dish for streak plating?

Answer 33

0,1,2,3,4 quadrants labeled on the bottom (gel-side up)

Question 34

Define colony in simple terms

Answer 34

a colony only contains the descendants of a single a sexually reproducing cell. all the cells in a colony are identical

Question 35

the growth habits/patterns of facultative anaerobes, aerobes, and anaerobes in broth

Answer 35

Another way of determining the pattern of growth of a microbe is by observing it grow in a thioglycolate tube. This medium is particularly designed to meet the oxygen requirements of different organisms (this will be discussed in more depth in Exercise 29). Microorganisms that are strict aerobes will grow only on the surface of the tube, forming a film on the surface. In this way, they ensure complete exposure to oxygen. Anaerobic microorganisms will tend to grow towards the bottom of the tube, where the oxygen tension is very low or zero. Facultative organisms (can grow in the presence or absence of oxygen), will grow all throughout the tube.

Question 36

the 3 different cultural characteristics of growth

Answer 36

form/shape elevation margin

Question 37

Before bacteria can be stained what must be made? Which staining procedures?

Answer 37

A smear preparation Gram staining, Capsule staining, Bacterial Endospore Staining, and Acid-Fast Staining

Question 38

What is smear preparation?

Answer 38

spreading/smearing bacteria onto slide, dry it, then heat fixed depends on the medium from which the bacteria will be removed however

Question 39

Purpose of heat fixing?

Answer 39

to make it adhere to the slide, to prevent it from washing off during the staining process.

Question 40

How to prepare smear from liquid culture (broth) and solid medium (agar slants and agar plates)

Answer 40

The first step in preparing a smear differs according to the type of medium from which the bacteria are removed. If the medium is a liquid (broth), one starts by placing a loopful of the broth directly onto the slide. If the bacteria are isolated from solid media (agar slants, agar plates), the first step is to place a drop of water onto the slide into which a small amount of bacteria is dispersed

Question 41

What would happen if the slide did not complete dry before you heat fix it?

Answer 41

you will end up boiling your microorganisms when heat-fixing

Question 42

Why do we need to perform a gram stain?

Answer 42

-It distinguishes between gram pos and gram neg cells

Question 43

What are the steps of gram staining?

Answer 43

Crystal violet is added to the smear first, followed by iodine which acts as a mordant. The iodine combines with the crystal violet to produce an insoluble precipitate inside the cell known as the crystal violet-iodine complex. In the next step, alcohol is added as a decolorizer. Observe cells under a microscope.The safranin (a counterstain) is added in the final steps of the Gram stain procedure and is taken up by the Gram negative cells. heat-fixed slide; use of crystal violet, iodine, decolorizer, and safranin

Question 44

What happens to the cell during staining? (ox. what happens to gram positive vs gram- negative cells when crystal violet is added to the slide)

Answer 44

-Gram positive cells will resist decolorization by the alcohol and retain the crystal violet-iodine complex. Gram negative cells are not resistant to the alcohol and lose all color in their cells. The safranin (a counterstain) is added in the final steps of the Gram stain procedure and is taken up by the Gram negative cells.

Question 45

What are the differences between gram-positive and gram-negative cells structures and exotoxin and endotoxin formation?

Answer 45

-Gram- neg cell walls contain a thin layer of peptidoglycan whereas gram-pos cells walls contain a thick layer of peptidoglycan. Gram positive and Gram negative bacteria produce exotoxins, only Gram negative bacteria produce endotoxins

Question 46

endo/exotoxin meaning?

Answer 46

Endotoxins - Inside lipids in bacterial membrane, generally released after cell death ● Exotoxins - Proteins exported from the cell

Question 47

What do gram-positive and gram-negative cells look like in the end?

Answer 47

-Gram positive cells appear bluish-purple due to their ability to resist decolorizing and retain the crystal violet-iodine complex. Gram negative cells will appear red as a result of the safranin counterstain.

Question 48

What would happen if you skipped a step during the gram stain procedure?

Answer 48

-Gram-negative organisms might not be visible, if you omitted the safranin step if you omit the Gram's iodine step while performing the Gram stain, gram-positive cells will most likely be pink in color. If you decolorize for more than 30 seconds, gram-positive organisms will most likely appear pink in color. if you forgot the crystal violet step while performing a Gram stain?Gram-positive and gram-negative organisms would stain pink

Question 49

Other types of responses from gram staining are gram variable and non-reactive. What do we mean by both?

Answer 49

Gram non reactives are bacteria without a cell wall such microbes, which do not stain or stain poorly; mycobacterium and various spirochetes.Gram variable bacteria possess the ability to appear both gram positive and gram negative on the same smear;

Question 50

What is a capsule?

Answer 50

-a substance that bacterial cells secrete that coat the cell and become the outermost mucoid layer not part of cell wall

Question 51

Why is a capsule useful for bacteria?

Answer 51

-The main function of the capsule is to protect the cell from drying out, barrier between cell and environment,Reserve of carbohydrates for bacterium, aid adherence to surfaces, aid avoidance of phagocytosis by immune systems)

Question 52

What is a capsule stain?

Answer 52

-Stain the background, leaves capsule unstained

Question 53

How do you define negative staining? Why is it used for capsule staining?

Answer 53

-Colors everything except the structure you wish to visualize because it is nonionic, it can't form bonds with dyes and cannot be stained. most staining techniques are based on the formation of an ionic bond between the stain and the membrane.

Question 54

How do you perform a capsule stain (names and purpose of the reagents)?

Answer 54

Place a drop of congo red on the slide and then add a sample from the bacteria specimen. Mix these and spread it on the slide so that a thin layer of this solution is formed on the slide. Air dry it. Do not heat fix it. Maneval's stain is then added to help dye the cell outline by helping the congo red penetrate through the capsule.

Question 55

What is an endospore ? What makes the endospores?

Answer 55

-Endospores are the dormant form of the bacterium that allow it to survive harsh environments that would otherwise kill the bacteria such as drying, starvation, increased temperature, and radiation. some endospores are formed as a normal part of the life cycle Vegetative cells produce endospores

Question 56

What is the Schaeffer-Fulton stain used for and how do you use it?

Answer 56

-Used to differentiate between an endospore and vegetative cell by using different stains. (Malachite green for endospore and saffranin for vegetative cells)

Question 57

Why do you steam the slides? for bacterial endospore staining

Answer 57

-Malachite green can only be formed by steaming the slides to force it into the spore coat. Use water to decolorize the veg cells while the spores retain the malachite green. Vegetative cells are counterstained with safranin

Question 58

Which of the gram-positive and negative bacteria could form endospores (remember the common genera)? Why are these genera important?

Answer 58

-Spores are primarily found as gram positive,rod-shaped organisms in the Clostridium and Bacillus genera. -They cause mad diseases (food poisining, anthrax, etc)

Question 59

Sporulation?

Answer 59

when a spore is produced by a bacterium. Not reproduction - sporulation leaves one vegetative cell and one spore ○ Spores can be used for identification: ■ Primarily found in gram+ rod cells (Clostridium, Bacillus) ■ Size and location - bigger or smaller than veg cell, located central, terminal, sub terminal

Question 60

1. What is an acid-fast bacterium (characteristics)?

Answer 60

-contains a large amount of lipids and waxes in their cell walls, primarily mycolic acid, which makes them relatively impermeable and resistant to many chemicals used as disinfectants -they are also resistant to desiccation, which allows them to remain viable in dried sputum for long periods of time

Question 61

Why do we need to take specific measures to dye them (chemicals for Ziehl-Neelsen Stain)?

Answer 61

-Because they cannot be stained by simple gram staining since they are resistant to it. Have to use different dyes.

Question 62

the steps in acid fast staining ?

Answer 62

-The Ziehl-Neelsen employs the primary stain carbol fuchsin -the stain is not taken up easily by the waxy cell wall, steam is used to drive it into the structure -The waxy cell wall of acid-fast prevents the primary stain from being removed by even the most potent decolorizes once it has penetrated -The acid alcohol decolozier used in the acid-fast stain will remove the carbol fuchsin from any cells that do not contain a thick, waxy cell wall. -A counterstain (Loeffler's methylene blue) is used to stain any non-acid fast cells in the smear. Acid Fast Stain Procedure: carbol fuchsi with heat breaks through the cell wall/lipoidal wall and turns the fast acid cells red. Then the smear is decoloirzed but the acid fast cells are reistant to the decolorizer, resulting in only the non acid fast cells being decolorized, leaving them colorless. The smear is then stained with methylene blue which only the decolorized/non acid fast cells absorb and turn blue while acid fast cells retain the red color.

Question 63

What bacterial species are acid fast bacteria and what diseases do they cause?

Answer 63

-Bacteria in the genus Mycobacterium which include M leprae and M. tuberculosis - the causative agents of leprosy and tuberculosis.

Question 64

What is the result for an acid-fast stain?

Answer 64

-The acid fast cells will appear read and the non fast acid cells will appear blue