lab practical 1

microscope 1

ocular

microscope 2

arm

microscope 3

stage clip

microscope 4

condenser knob

microscope 5

course adjustment knob (10x)

microscope 6

fine adjustment knob

microscope 7

revolving nose piece

microscope 8

objective lens

microscope 9

condenser

microscope 10

stage

microscope 11

stage adjustment knobs

microscope 12

diaphram

microscope 13

lamp

microscope 14

base

coccus

bacillus

spirillum

Why negative staining

When bacteria do not stain well with other methods. Quick not heat fixing. Bacteria negative and so is stain so repel and satin background

What does a negative stain look like?

Why simple stain

 View bacterial size, shape, or arrangement. Bacteria are negative, stain positive attract and stain bacteria. Must be heat-fixed to kill bacteria and adhere them to the slide.

What does simple stain look like

why gram stain

differential stain because two groups of bacteria with different cell walls. Gram +: purple. Gram -: pink.

What does gram stain look like

why acid fast stain

some bacteria don’t stain well with typical stain techniques. Uses heat. Drives stain into bacteria through the thick waxy lipid present in cell wall. Acid fast: red. Not acid fast: blue

what does acid fast look like

why endospore staining

some bacteria produce resistant dormant structures. Endospores are difficult to stain by ordinary methods. The location of endospores in cells can help identify bacterial species. Endospores green. Pink normal cell. Pink with green: vegetative cell with endospore

 

What do endospores look like

Why Capsule staining

Bacteria with sticky sugary layer around. Combination of negative stain and simple stain. Capsule clear, cell pink, background black

What do capsule stain look like

pipette a

air

pipette e

expel

pipette s

suction

temperature what is being affected

the enzymes or proteins denature with too low, also membrane fluidity

membrane becomes too fluid if temp is too low

thermoduric bacteria survive why

form endospores

what is thermoduric

bacteria that do not grow at elevated temperatures.

Psychrophiles

0-20 degrees Celsius

Mesophiles

20-45 degrees Celsius

Thermophiles

55+ degrees Celsius

Why do bacteria have minimum and maximum growth pH

The proteins and enzymes denature and hydrogen bonding is effected

Acidophiles

0-5.5 pH

Neutrophiles

5.5-8.0 pH

Alkalophiles

8.0-11.5 pH

Hypotonic

Water move into cell

Hypertonic

Water move out of cell

Isotonic

No net gain or loss of water

Facultative halophile

0-10% NaCl

Obligate halophile

15+% NaCl

Obligate aerobe

Grow only in presence of O2

Facultative anaerobe

Organisms grow with or without presence of O2, but prefer it

Obligate anaerobe

Killed by O2

Aerotolerant anaerobes

Don’t use O2 for growth and not harmed either

Microaerophiles

Less than atmospheric concentrations of O2

oxygen growth 1

obligate aerobe

oxygen growth 2

facultative anaerobe

oxygen growth 3

obligate anaerobe

oxygen growth 4

aerotolerant

oxygen growth 5

microaerophile

Bacteriostatic

Antibiotics inhibit bacterial growth

Blurred line

 

Bacteriocidal

Antibiotic kills bacteria

Solid line

Some people do not wash their hands correctly and don’t remove all the transient microorganisms present. What could be some problems with their techniques? b

Missing spots, not enough time, not using soap 

What is a healthcare associated infection (nosocomial infection)? How are they usually spread (be specific)? 

It is an infection that is acquired in a clinical/healthcare setting that is most commonly spread by healthcare workers who don’t properly wash their hands. 

Are hand sanitizers a safe alternative to washing hands with soap and water (explain)? 

Yes but it doesn’t work completely depending on how dirty your hands are and if there is organic matter present. It also kills your normal flora. 

What is the different between cleaning and disinfecting? 

Cleaning removes dirt and some of the bacteria while disinfecting destroys vegetative (non-endospore forming) pathogens 

What physical or environmental factors can influence the activity of a disinfectant? 

Organic Matter, Surface Type, and time 

What is the difference between a disinfectant and an antiseptic? What is an example of each? 

A disinfectant kills bacteria on a nonliving surface. Examples include Lysol and bleach. An antiseptic kills bacteria on a living surface and includes hydrogen peroxide and alcohol. 

 

What is a hot zone in your home that requires disinfection? Why is it considered a hot zone? 

The kitchen and bathroom are considered hot zones because there is a significantly higher amount of bacteria there 

When doctors wash their hands for surgery, they must scrub their hands with a brush. What category of bacteria are surgeons trying to remove by using a brush? 

They are trying to remove their normal flora 

What is the difference between resolution and magnification? 

Magnification makes the image or cell appear larger while resolution provides more detail of the image 

The 40X lens is called the ‘high dry’ lens. Why do we call it that? 

It is the highest objective our microscopes have without adding oil 

Define parfocal. How does this characteristic affect the way you would view a slide? 

When you switch from one objective to another, the object stays in the same plane of view so all you have to do is adjust the fine adjustment knob. 

What is the function of the condenser? The iris diaphragm knob?

The condenser concentrates the light and the iris diaphragm controls light intensity 

 

What are the two functions of the ocular (eyepiece) lens? 

The functions are to magnify and measure 

In this course, which objective lens will be used the most? Why? 

White will be used to look at shape and color which is the oil immersion lens 

If you are using the oil immersion lens to view your bacteria, what is the total magnification? What does this measurement mean? 

1000X, the cell will appear 1000X larger 

Why must we use immersion oil to get good resolution with the 100X lens? 

To get more light into the objective lens and to trap the light 

 

If 50 ocular micrometer divisions = 5 stage micrometer divisions and if one division of the stage micrometer = 0.1 mm. The cells you measure are 42 ocular micrometer divisions long. Therefore the cells are _____ mm long. 

.42 mm 

Before the microscope is put away, name 2 things you must do. 

You must wipe off any oil, wrap up the cord, and put it on the lowest objective lens 

Why don’t acidic or background dyes stain the bacteria? 

Both bacteria and dye are negative so it is repelled 

Why do basic dyes stain the bacteria? 

The dye is positively charged and bacteria is negative so they are attracted 

When using a basic stain, the cells must be heat fixed. What are the 2 benefits of heat fixing? 

It is used to kill the bacteria and adhere the bacteria to the slide 

What are the benefits of using a simple stain? 

The stain highlights the entire microorganism to show shape and size. It is also simple and fast 

What are the benefits of using a negative stain? Why/when would you want to do a negative stain instead of a simple stain? 

No heat is used so it keeps the shape and size 

For the Gram stain, name the reagent used for each and its purpose. Also identify the color after each step. 

Primary stain-crystal violet stains everything purple so both + and – are purple 

Mordant-iodine shrinks the pores in the cell wall of + to trap dye but both + and – are purple 

Decolorizer-acetone alcohol removes excess crystal violet so + is purple and – is clear 

Counterstain-safranin stains anything that is not already stained pink so + is purple and – is pink 

 

Why must cultures between 18-24 hours old be used for the Gram stain? 

When the cells are older the pores in + cells are more likely to be weak and leak which makes them unable to hold the dye. This would create false negatives 

What is the step that is the most likely to cause poor Gram stain results. Describe, 

Decolorizer as it is easy to be left on too long and then pull dye out of the gram + cells 

What cell structure is the most important for a Gram stain? Why (be specific)? 

Cell wall because of the thickness; the + have a think wall while – has a thin cell wall and outer membrane 

What is the difference between virulence and a virulence factor? 

Virulence is the severity of disease (how harmful it is) and virulence factor is the component of bacteria that gives it the ability to cause disease 

What makes a bacterium acid fast? 

There is a waxy lipid layer of mycolic acid which allows it to resist drying and certain disinfectants 

Why must the bacteria be heated with the stain in the acid fast procedure? 

To drive/force the stain into the mycolic acid lipid layer 

What are 2 diseases that can be diagnosed using the acid fast stain? 

leprosy and TB 

What is an endospore? Why does bacteria produce endospores? 

Resistant, dormant (non-living) that has a durable coat around the DNA that is created in unfavorable conditions 

How can an endospore be a virulence factor? 

it can survive harsh conditions and preserves the bacteria’s genetic material. It lives in unfavorable conditions such as high heat, household disinfectants and can live 100s of years in dormant state 

What are 2 diseases can be diagnosed using an endospore stain? 

Anthrax, Botulism, and Tetanus 

Of what value is the capsule to a bacterium? 

Sugary/sticky layer that help it stick to the host. The body does not recognize it as foreign because of the sugar so the bacteria can hide from the white blood cells. The capsule is also a back-up food source for a bacteria. 

Why does the capsule stain require both a negative stain and a basic stain? 

The negative stain makes the background black, the basic stain makes the cell purple/pink, ad the capsule remains clear 

What type of infections are generally caused by bacteria that have a capsule? 

Respiratory 

What causes the difference between nutrient media broth, nutrient media semi-solid and nutrient media solid? 

The amount of agar 

Lauria Bertain Agar (LBA) is used in lab for most experiments. The recipe is: 10g Tryptone, 5g Yeast Extract, 10g NaCl, 15g agar, 1L distilled water. Is this complex or chemically defined? How do you know? 

Complex; there is yeast extract and extract or digest are not specific so it is not chemically defined 

If you wanted to sterilize 3L of LBA how would you do it? 

Autoclave at 121 degrees C with 15 PSI for 20 min 

If you wanted to sterilize a liquid sample of heat labile antibiotic, how would you do it? 

Bacteriologic filter