BIOL 3401 General Microbiology Laboratory Fall 2020

Brightfield Microscopy

Terms to know
Brightfield microscope
Magnification
Interocular distance
Working distance
Resolution
Parfocal and parcentral

• Different kinds of microscopes include darkfield, fluorescence, phase-

contrast and brightfield.

• Brightfield is different from other kinds of microscopy. It allows light to pass

directly to the eye without being deflected by an intervening opaque plate in
the condenser, resulting in objects appearing dark against a bright
background.
• How to carry a microscope
▪ Make space in your work area.
▪ Make sure the cord is nicely wrapped before picking the scope up.
▪ Carry the microscope using one hand under the base and one hand
around the arm.
▪ Put it down gently.

• Parts of a microscope- important to know all these parts

▪ On/off switch
▪ Base
▪ Light source
▪ Condenser
▪ Diaphragm
▪ Mechanical stage
▪ Objectives
▪ Nosepiece
▪ Oculars or eyepieces
▪ Head
▪ Diopter adjustment ring
▪ Arm/neck
▪ Fine adjustment knob
▪ Course adjustment knob
▪ Stage adjustment knob
 BIOL 3401 General Microbiology Laboratory Fall 2020
2020
ANSWER THE FOLLOWING QUESTIONS OR FILL IN THE BLANKS:

1. How many lens systems does a compound microscope have?

a. All compound microscopes have 3 lens systems: Oculars, the
objectives, and the condenser.

2. What is the magnification of ocular lenses?

a. The ocular lens, at times, will have a magnification of 10X

3. What is the magnification of the objective lenses?

a. Magnification of objective lenses on most laboratory
microscopes are low power 10x, high dry magnification 40x,
and total magnification 100x.

4. How do you determine the total magnification of an object?

a. The magnification of an object is determined by multiplying the
power of the ocular lense times the power of the objective lens.

5. If 100x objective is in place, what is the total magnification of the specimen in

place?
a. 10 x 100 = 1000 total magnification

• Steps for obtaining the best image

• Start with the 100X objective or low-power objective, if scanning is
not available, using the fine adjustment focus knob. Fine focus to
make the image sharper DO NOT READJUST THE COURSE FOCUS
KNOB.

• Adjust interocular distance and diopter adjustment. Adjust the light

settings to get the best image.

• Change the objective lens for next higher magnification, by

rotating that nose piece____ .

• Use the fine adjustment focus knob for sharper image.

• Repeat steps 5 and 6 until you have visualized your slide with the
40x objective.
• After you are confident that you visualized microorganisms at 40x,
you are ready to move to 100x or oil immersion objective. To
do so, hover the objectives between 40x and 100x by rotating the
nose piece and place a single drop of immersion oil on the slide.
Gently rotate the nose piece to place 100x objective on place.
BIOL 3401 General Microbiology Laboratory Fall 2020

• Use the fine adjustment focus knob for sharper image.

13. Resolution or resolving power of a lens is its ability to

Reach the least distance between points in the object that can be distinguished in the image.
BIOL 3401 General Microbiology Laboratory Fall 2020
2020

▪ Numerical aperture is a mathematical expression that describes how

the condenser lens concentrates and focuses the light rays from the
light source.
▪ Its value is maximized when the light rays are focused
onto a cone of light that then passes through the specimen into
the objective lens
▪ diminished 15 when refracted light rays are lost

16. Please outline the steps to correctly put away a microscope.

1. Remove the slide from the stage.

2. If immersion oil has been placed, wipe it off the lens and stage with lens
tissue. Also , make sure that no immersion oil is on the 40X objective. (Do
not wipe off oil off slides you wish to keep. Simply put them into a slide box
and let them oil drain off.)
3. Rotate the lower-power objective into position.
4. If the microscope has been inclined, return it to an erect position.
5. If the microscope has a built-in movable lamp, raise the lamp to its highest
position.
6. If the microscope has a long attached electric cord, wrap it around the base.
7. Adjust the mechanical stage so that it does not project to far on either side.
8. Replace the dust cover.
9. If the microscope has a separate transformer, return it to its designated
place.
10. Return the microscope to its correct place in the cabinet.
BIOL 3401 General Microbiology Laboratory Fall 2020

Culture Media Preparation

Terms to know
Media
Medium
Complex, defined, selective and differential media

• Media, singular culture , is an artificial growth mixture used to

cultivate/grow microorganisms. It supplies the microorganisms with all
the nutrients _an energy requirements _necessary for growth.

• Consistency-
▪ Solid ( 1.5 % agar; seaweed derivative, complex
polysaccharide energy/food)
▪ Semi-solid (0.4% agar, softer for motility studies tests)
▪ Liquid- Fermentation studies, indole utilization , large volumes, tests
such as MR-VP

• Seven basic nutritional requirements necessary for all cells to grow

▪ Note- You should be able to know/list the seven basic nutritional

requirements provided in the media and understand that formulating
and choosing a medium for a particular microorganism is a
complicated process.

▪ H2O
▪ DI water has to be used minerals in the tap water might
interfere with components of the media.

▪ Carbon- in general, based on C sources

▪ Autotrophs can fix carbon dioxide
▪ Heterotrophs use organic molecules as a C source such as
polysaccharides, amino acids, peptides and proteins. Meat and
plant extract are added to complex media to supply these
nutrients

▪ Energy- based on energy source

▪ Chemoorganotrophs- breakdown of organic molecules by
respiration or fermentation

▪ Chemolithotrophs- oxidize inorganic ions such as nitrate or

iron
BIOL 3401 General Microbiology Laboratory Summer II 2020

▪ Photoautotrophs- solar energy into chemical by

photosynthesis.
• Most bacteria are not photosynthetic.
• Ex. cyanobacteria and green and purple sulfur bacteria

▪ Photoheterotrophs- derive energy from photosynthesis but

carbon needs come from organic molecules such as succinate
or glutamate

▪ Nitrogen-
▪ Component of nucleic acids and proteins
▪ Can be used in respiration, nitrogen fixation

▪ Trace mineral- small amounts,

▪ Enzyme cofactors, vitamins,
▪ Ex. sodium, potassium, calcium, magnesium

▪ Growth factors and vitamins-

▪ Trace amounts

▪ Hydrogen ion concentration-

▪ acidophile (acid-loving) and alkaliphiles (base-loving)

• Types of media
▪ Defined vs. complex
▪ What is defined media? How is it different from complex media?
▪ What is selective media? How is it different or the same as
differential media? [Note: we will have an entire lab dedicated
to media. For now, just know the definitions.]
• Process to make media plates
Note- I just want to emphasize on these general steps, we do not have to
go into much details. Please watch the YouTube video “Making Microbiological
Media” to see the process of making media.

▪ Sterilization- autoclaving at 15 psi, 121° C, 20 minutes. Kills vegetative

cells and spores.
▪ Aseptically pour the media in sterile petri plates, after is has cooled
down but not enough for agar to solidify.
▪ Allow to cool down.