1) contrast to see. Light microscope magnification

1)   
Explain the general concept of
how a brightfield microscope works.  What
are the major limitations? (1pt)

 

Brightfield microscope working
procedure;

 

In microscopy after
locating a sample on the stage, light comes from the light source . There is a
lens which is named as condenser aimed the sample.  The condenser has a diaphragm which has
aperture. this diaphragm is for controlling and focusing the light on the
sample. Light passes through the sample. this light is collected by an
objective lens. This lens is on the stage. The objective helps the light more
visible. The user can see the sample from oracular lens or eyepiece. The
contrast, which is
absorbed by stains, pigmentation, or dense areas by light, allows
you to see the sample.

 

Major limitations are;

 

Some
biological samples have low contrast to see. Light microscope magnification is
around 1300X. There are bigger magnifications but if you increase it, the
image’s clarity will decrease. Naturally colorless and transparent samples are
visible. They should be stained before using the brightfield microscopy.

 

 

2)   
What is the Refractive
Index?  What is the working distance? (0.5
pt)

 

Refractive index is the ratio of the velocity of light in
a vacuum to its velocity in a specified medium. For calculation the
working distance, Numerical aperture is used. Numerical
aperture is basically for how much good detail you can see. The numerical
aperture formula is

 

Numerical Aperture (NA) = ? • sin(?)

 

a = one-half of the objective’s opening angle 

? = refractive
index of the immersion medium

           

            Refractive index is the limiting
factor to achieve numerical apertures more than 1.0. So, to get bigger
numerical apertures, the refractive index must be increased.

 

 

3)   
What is the total
magnification when using the 4X, 10X, 40X, and 100X objectives? (0.5pt)

 

Total magnification is calculated by multiply the objectives (4X, 10X, 40X,
and 100X) and power of eyepiece (10X)

 

Total
Magnification;

(4X)
* (10X) = 40X

(10X)
* (10X) = 100X

(40X)
* (10X) = 400X

(100X)
* (10X) = 1000X

 

4)   
Bacterial species are rarely
identified by colony morphology alone. 
Explain why this is usually the case. Give one or more example(s) of
other techniques that may aid in identifying an unknown microorganism. (1pt)

Bacterial morphology deals with size, shape, and chromogenesis, opacity, elevatin,
surface. margin generally, these criteria may be enough to identify the
species.

Also Aggregation and
biochemical testing are techniques to identify the microorganisms.

 

5)   
Define differential stain. How
is this technique different from a simple stain? (0.5pt)

 

Differential
stain works with chemical as simple stain. In this technique colors are
important as simple stain. But there are more than 3 chemicals. The differential
stain does not give the same color to the bacteria. Differential stain is for
identify the cell types, their structure basis on their different colors.

 

6)   
Compare and contrast cell
walls of G+ and G- bacteria. (0.5pt)

 

·      Gram+ bacteria has thick (multilayered) peptidoglycan layer. Gram- bacteria
has thin (single-layered) peptidoglycan
layer.

·      Gram+ bacteria has not a outer membrane. Gram- bacteria has a outer
membrane.

·      Two of them has a cytoplasmic membrane.

·      Gram+ bacteria has low lipid and
lipoprotein content. Gram- bacteria has high lipid and lipoprotein content.

·      Gram+ bacteria is More susceptible to
antibiotics.

Gram- bacteria is More resistant to antibiotics.

 

 

7)   
Make a table with the color of
E. Coli and S. Epidermidis after each step you completed during your Gram
stains in lab (Crystal Violet, Iodine, Alcohol, Safranin). (1pts)

 

E. Coli

S. Epidermidis

Primary Stain

Crystal Violet

Purple

Purple

Mordant

Iodine

Purple

Purple

Decolorizer

Alcohol

Colorless

Purple

Counterstain

Safranin

Pink

Purple

 

 

 

“Brightfield
Microscopy – Uses & Advancements; Microscope Reviews; Pros and Cons.” MicroscopeMaster, www.microscopemaster.com/brightfield-microscopy.html.

 

“Bright-Field
microscopy.” Wikipedia, Wikimedia
Foundation, 19 Jan. 2018, en.wikipedia.org/wiki/Bright-field_microscopy.

 

“Refractive
index.” Wikipedia, Wikimedia
Foundation, 29 Jan. 2018, en.wikipedia.org/wiki/Refractive_index.

 

“Properties
of Microscope Objectives.” Nikon’s MicroscopyU,
www.microscopyu.com/microscopy-basics/properties-of-microscope-objectives.

 

http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html

 

“Numerical
aperture.” Wikipedia, Wikimedia
Foundation, 31 Dec. 2017, en.wikipedia.org/wiki/Numerical_aperture.

 

“Understanding
Your Objective.” Thermo Fisher Scientific, www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/fundamentals-of-fluorescence-microscopy/understanding-your-objective.html.

 

 

http://www.cas.miamioh.edu/mbiws/microscopes/Magnification.html

 

Libretexts.

“8: Bacterial Colony Morphology.” Biology LibreTexts, Libretexts, 2 Jan. 2018,
bio.libretexts.org/Labs/Microbiology_Labs_I/08%3A_Bacterial_Colony_Morphology.

 

“Chegg.com.” Definition of
Bacterial Morphology | Chegg.Com, www.chegg.com/homework-help/definitions/bacterial-morphology-14.

 

“Bacterial
cellular morphologies.” Wikipedia, Wikimedia Foundation, 22 Jan. 2018,
en.wikipedia.org/wiki/Bacterial_cellular_morphologies.

 

http://www.nios.ac.in/media/documents/dmlt/Microbiology/Lesson-11.pdf

 

drillmaster2001.

“What is the difference between a differential and a simple stain
Differential.” Simple stain
Differential,
www.coursehero.com/file/p18b4vm/What-is-the-difference-between-a-differential-and-a-simple-stain-Differential/.

 

“Gram-Positive
vs Gram-Negative Bacteria.” Gram-Positive
vs Gram-Negative Bacteria – Difference and Comparison | Diffen, www.diffen.com/difference/Gram-negative_Bacteria_vs_Gram-positive_Bacteria.