8-Acid-Fast Stain_needs Qs (2)
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8 - Acid-Fast (Ziehl-Neelsen) Stain
Objective/ purpose/ learning goals: After performing this lab exercise, students will be able to a.
explain principles of acid-fast stain
b.
perform acid-fast staining procedure
c.
differentiate bacteria into acid-fast and non-acid-fast groups based on their appearance under microscope
Key terms
Carbol fuchsin, acid-fast stain, non-acid-fast stain, mycolic acid
Introduction / theory/overview: The Acid-Fast stain is a differential staining technique that was first developed by Paul Ehrlich in 1882 and later modified by Franz Ziehl and Friedrich Neelsen in 1883. Therefore the acid-fast stain is also called the Ziehl-Neelsen stain
. The acid-fast stain differentiates bacteria into acid-
fast and non-acid-fast groups. This method is used for members of the genus Mycobacterium and
Nocardia. Acid-fast microorganisms are resistant to simple or Gram staining methods. Principle of Acid-Fast Stain:
As the dye carbol fuchsin is applied to a bacterial smear, it solubilizes the lipid material present in the bacterial cell wall. With application of heat, carbol fuchsin further penetrates through the lipid-wall and enters into the cytoplasm. At this point all cells appear red. When these red cells are decolorized with acid-alcohol decolorizing agent (3% HCL in 95% alcohol), the acid-fast cells are resistant to decolonization because of the presence of large amounts of mycolic acid (a specific kind of lipid) in their cell wall, which prevents the penetration of decolorizing solution. The non-acid-fast bacteria lack mycolic acid in their cell walls such that they are easily penetrated by the decolorizing agent and thus decolorized. This results in colorless cells. The smear is then counterstained with methylene blue. Only decolorized cells will absorb the counterstain, taking up its color and appear blue. Acid-fast cells do not absorb methylene blue and retain the red color.
Video Links for Principle and Procedure of Acid-Fast Staining: https://www.bing.com/videos/search?
q=principle+of+acid+fast+stain+video&docid=608050395440549737&mid=354ADBE14A8139
83005F354ADBE14A813983005F&view=detail&FORM=VIRE
https://www.bing.com/videos/search?
q=principle+of+acid+fast+stain+video&docid=608009060688789857&mid=06E0DA9793E0A
B04151006E0DA9793E0AB041510&view=detail&FORM=VIRE
https://www.bing.com/videos/search?
q=principle+of+acid+fast+stain+video&docid=608046706067705648&mid=9A6A60A0D590A
770CDF99A6A60A0D590A770CDF9&view=detail&FORM=VIRE
Materials, equipment and organisms (per student/ per group): a.
Live Organisms: Mycobacterium pheli, E.coli
b.
Two slides per student for smears c.
Standard materials used for making, drying and heat-fixing smears, d.
Staining hot plate, Carbol fuchsin dye, acid alcohol decolorizing solution, and filter paper. Procedure: a.
Obtain a slide and make two smears, one for Mycobacterium pheli
and one for E. coli
on
the slide. Take time to break the clumps of Mycobacterium
when making smears before drying your smear on designated hotplate. Heat-fix the smears. Each student will make at least two slides, each with smears for both organisms side by side.
b.
Find the fume hood where acid-fast staining materials are located
c.
Put heat fixed smears onto staining hot plate in the fume hood.
d.
Place filter paper(s) on the heat fixed smear and use the dropper to completely saturate the filter paper with carbol fuchsin
dye.
e.
Allow dye to stain your smear for at least 5 minutes.
f.
Wipe off the bottom of the slide with a wet paper towel and rinse off the dye with tap water.
g.
Decolorize carefully with acid alcohol (3-5 drops) for a minute and rinse your slide with water.
h.
Counterstain with methylene blue for a minute.
i.
Rinse off the counter stain, blot dry ( and place coverslip if required by your instructor).
j.
View your slide at 100X under oil
Safety and disposal:
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Related Questions
a. How was the specimen prepared for the microscopy technique applied? (for e.g. stained with H&E stain, Gram stain, unstained)
Â
b. What is the microscopy technique and magnification used to obtain this image?
Â
c. What is the basic principle of image formation using this microscopy technique?
Â
d. What can be observed and concluded from the image of the specimen?
Â
e. Are there any potential aberrations present in this specimen image? Describe these and how they may affect interpretation of the result.
arrow_forward
Discuss about Fluorescencemicroscopy
arrow_forward
Direction: Read and analyze the following laboratory experiment and answer the
following question.
PART 1: SURFACE AREA AND CELL SIZE
Materials: Agar containing NaOH, and the pH-indicator dye phenolphthalein
cured into cubes of various size, 3 plastic cups, HCl, metric ruler, paper towels.
Methodology:
1. Safety: Wear goggles and nitrile gloves while completing this lab.
2. Obtain three different size blocks of pink or blue agar. Using a ruler,
measure the length, width, and height of the three blocks given below. Cut
the agar according to the given dimension.
Small = 1 cm x 1 cm x 1 cm
Medium = 2 cm x 2 cm x 2 cm
•
• Large = 1 cm x 1 cm x 6 cm
3. Record your data.
4.
Pour HCl or vinegar into two small cups. Place the one larger "cell" into one
cup and the two smaller cells in the other cup. Start timing 30 minutes.
5. After 30 minutes, remove the cells and blot them dry with a paper towel.
6. Using your ruler, measure the distance the HCl has diffused into the blocks
as shown on the…
arrow_forward
Please include your reference below for my further research. Thank you!
1. What are the basic components of a Fluorescence Microscope and what are the functions of each? Â
2. Are there any parts that you can remove without compromising accuracy and utility of the equipment?Â
3. Can you suggest additional components to improve the equipment?
arrow_forward
Subject : Environmental MicrobiologyÂ
Can u use the information given below to answer these 2 questionÂ
1.Provide an aim for this lab
2. Provide objectivesÂ
DISCUSSION QUESTIONS
Â
What is the relationship between the resolution power and the useful magnification that may be obtained with the light microscope?Â
What determines the resolving power of the lens system?               Â
What is the limit of resolution obtainable with the light microscope?                        Â
How you will distinguish between bright field and dark-field microscopy and provide a specific example where each would be method of choice for observing a culture of bacteria? Â
What advantages does electron microscopy have over light microscopy? Â
What are disadvantages of electron microscopy over light microscopy?               Â
#Compare the use and the methodology of TEM with SEM? Provide at least one example where each would be the method of choic
arrow_forward
Copy and paste the link below and watch the video on Youtube
https://www.youtube.com/watch?v=8RBs0Ghg_48
Â
Answer the following Questions:
1. What are the chemicals and materials used in gel electrophoresis?
Â
2. Draw a schematic diagram of a gel electrophoresis set-up
3. Describe the procedure in doing a gel electrophoresis experiment. Why is there a need for a leveling bubble/leveler? What is the use of the rubber dam?
Â
4. What is the use of ethidium bromide and why must you wear gloves when you handle it?
Â
5. What makes the DNA fragment move towards the positive plate?
Â
6. What is the purpose of glycerol in the sample buffer?Â
Â
7. What is the use of a DNA ladder?Â
Â
8. What will happen when you increase the voltage of the set-up?
Â
9. Can gel electrophoresis be used to separate amino acids? If so, how is it done?
arrow_forward
topic: gel electrophoresis
. What are other staining methods that can be used for PAGE?
arrow_forward
Make a concept map/flow chart for this technique (Cellulose Tape Perianal Swab)
arrow_forward
MICROBIOLOGY:Â Microscopic Morphology of Microbes
Write your introduction (This includes principles, significance of the study, objectives of the experiment and how the objectives were achieved. This part must also be in the passive voice and past tense. Introduction must be short but packed with relevant content).
another:
What is the advantage of the Gram stain over the simple stain?
What is the theory about the mechanism of the Gram-stain reaction?
arrow_forward
3) Define gel electrophoresis, including its theory and application. Describe the steps of
running gel electrophoresis using the following image. More detailed reading:
https://www.sciencedirect.com/topics/medicine-and-dentistry/agar-gel-electrophoresis
POWER SUPPLY
CATHODE
ELECTROPHORETIC
BUFFER
ANODE
WELL
SAMPLE
AGAROSE
GEL
POWER SUPPLY
CATHODE
ANOCE
HIGH MOLECULAR
WEIGHT SPECIES
LOW MOLECULAR
WEIGHT ANALYTES
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please help me make this procedure a flow chart
Â
III. Procedure: A. MicroscopyOperating Procedure:Place the microscope close to the edge of the table. Select a suitable stool so that when looking into the eyepiece, your back is straight and your neck is bent at the nape.1. Lower the body tube by turning the course focus knob until the 10x or 16mm objective reaches the downward stop.2. Look through the eyepiece and adjust the mirror to the position which provides the brightest and the most evenly illuminated field of vision which is the circular area seen in the eyepiece. Raise the condenser until its top lens at the same level as the stage. Place the slide on the stage and fasten it using the stage clips.3. Position the specimen area of the slide over the center of the stage aperture.4. Looking through the eyepiece, raise the coarse focus knob until the image appears. Focus as sharply as possible. Low power objective has a much greater depth of focus and is generally used for the…
arrow_forward
Describe your results in the following data
chart as directed in the instructions for
the laboratory exercise.
Treatment
Control
0.01% Bleach
0.1% Bleach
1.0% Bleach
2.5% Lysol
5% Lysol
10% Lysol
0.03% Hydrogen
Peroxide
0.3% Hydrogen
Peroxide
3.0% Hydrogen
Peroxide
10% Isopropyl
Alcohol
30% Isopropyl
Alcohol
50% Isopropyl
Alcohol
Growth
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Question:-
Starting with data collection, describe the steps necessary to determine a 3-Dimensional structure using electron microscopy methods.
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1:07
< Ð’ack
Untitled 6
Brownian movement of bacteria:
O a. Can be observed by the of soft agar technique
O b. Can be observed by staining of Staphylococcus aureus
Oc. Is common in Escherichia coli or Proteus vulgaris
O d. Is common in Staphylococcus aureus
O e. Can be observed at the edge of a microscope
CLEAR MY CHOICE
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Choose the one answer that fits best. Which of the following statements regarding the proper procedure for using Micropipettes is NOT correct?
O a. You cannot use a 2-20 µl micropipette to pipet 200 µl
O b. To expel all the liquid from the tip, you have to press the eject button
O c. To draw up solution, press and hold the plunger at the first stop before entering the solution
O d. Micropipettes always require the use of a disposable plastic tip
O e. While pipetting, micropipettes should always be held as straight as possible
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PROCEDURE:
1. Set two pencils down parallel from each other. Make them about 2-3 inches apart as the length of your slides to keep
things easy.
2. Stick a long piece of tape over the
two pencils and to the table on either
side of the pencils to hold the tape
tightly between the two pencils like
a bridge.
3. Don’t touch the sticky side of the
tape or you will ruin the microscope.
Drop a small drop of water onto the
top of the tape using the pipette or
medicine dropper.
4. Make 3-4 lines of tape and add a
different-sized drop to each one.
This will help determine what size
of water droplet produces the biggest
magnification.
5. Prepare a rectangular shape of plastic cover. Put a small slice of onion.
Slide the rectangular shape of the plastic cover with the small slice of onion under the pieces of tape and observe the size
of the onion on different droplets. Write your observations on the table below.
No. of drops
Observation
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mead.
Post Lab #2
Smear & Stain Preparation
Name: Leidiawa MontaNo
Date:
1. Why are thick or dense smears less likely to provide a good smear preparation for
microscopic evaluation? Please explain.
Becapse, it will diminish the amount ds latcan Pass through by mam
1t difficult to See under the micioscol e, s less liket to Prowde a go
image.
2. What could potentially happen if you leave the slide exposed for too long to the open
flame? Why do you have to be careful?
we can form ring Patterns if we expose the slide for ta0
the flame
3. During the preparation of a smear leading into simple staining of the bacterial culture
S. epidermidis you forgot to heat fix the slide. What would you see on this slide as
compared to a slide that was properly prepared? Please explain.
4. You partner stained bacterial cells and saw only the background and not the actual cell
was stained. Your partner thought this was a mistake. Please explain what type of
staining method this is, how it works and why the…
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Gel used in electrophoresis is/are:
a. Agarose gel
b. Polyacrylamide plain gel
c. Polyacrylamide SDS
impregnated Polyacrylamide gel
(Sodium dodecyl sulphate)
arrow_forward
Discuss SSD (non-isocentric) technique and SAD (isocentric) technique including the advantages and disadvantages of each type of setup.
arrow_forward
Discuss the steps from dissection of tissue to producing a section on a slide ready for staining, as provided for this practical and as would be carried out in a hospital lab. include principles and procedures for each step. maximum word limit 400
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A. Purpose:
Figure 1
B. Materials:
Microscope
Magazine
Slides and cover slips
Paper towels
Pipette
Scissors
C. Procedure:
1. Careful carry a microscope to your lab area. Make sure to hold it with one
hand under the base and one hand on the arm as shown in Figure 1.
2. Plug the microscope in and turn it on. Take a moment to look at all the parts of the microscope. Then look
at your ocular lens. What is the magnification of the ocular lens (eye piece)?
Figure 2
3. Fill in the chart to show the total magnification for each objective lens.
Magnification
of Ocular Lens
Magnification of
Objective Lens
Objective Lens
Total
Magnification
Low Power
Medium Power
High Power
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Horizontal sequence :VIRL
Vertical sequence:MKF
Â
Scoring rules: g/o = -3, g/e = -1, match or mismatch - from PAM250 substitution matrix below.Â
SW algorithm.Â
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1. Complete the scoring matrix.Â
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Scoring matrix with PAM250 scores:
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2. Set up, initialize and complete the SW matrix.Â
3. Retrace, align and score alignment(s).      Â
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Align and score all optimal alignments here.Â
PLZÂ the arrows and circles for the matrix and path(s) AND SHOW ALL possible Alignment
arrow_forward
Horizontal sequence :VIRL
Vertical sequence:MKF
Â
Scoring rules: g/o = -3, g/e = -1, match or mismatch - from PAM250 substitution matrix below.Â
Â
SW algorithm.Â
Â
1. Complete the scoring matrix.Â
Â
Scoring matrix with PAM250 scores:
Â
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R
L
M
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F
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2. Set up, initialize and complete the SW matrix.Â
3. Retrace, align and score alignment(s).      Â
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Align and score all optimal alignments here.
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B. Write the function/s of each part of the microscope listed below.
a. Eyepiece
b. Draw tube
c. Hemispheric prism housing
d. Dust shield
e. Revolving Nose Piece
f. Objectives:
Scanner
LPO
HPO
ΟΙΟ
g. Arm
h. Coarse Adjustment knob
i. Fine adjustment knob
j. Slide holder & clip
k. Rear knob
I. Front knob
m. Central aperture
n. Condenser
o. Iris diaphragm
p. Mechanical Stage
q. Mirror
r. Mirror rack
s. Stage adjustment knob
t. Base
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The significance of using immersion oil when using 1OOX objective lens is
The significance of using immersion oll when uskng 100X objective lens is:
It will increase the contrast
by having same refractive index as the lens, loss of light by refraction, reflection
and diffraction is minimized.
the specimens need not be killed, fixed and stained
more light is captured for better illumination, allowing the specimen to fluoresce
i.
il.
iii.
iv.
A light microscope should have the following features
contrast
Resolution
magnification
fluorescence
ii.
iv.
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Question:-
Describe control strains used in the clinical microbiology laboratory and explain their maintenance in the laboratory. ( write BY WORD and all steps I need).
Introduction
Discussion
References
arrow_forward
Discuss the principles and applications of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry in molecular diagnostics.
arrow_forward
Explain the importance of steam during the acid-fast staining
procedure.
Discuss the advantages of differential staining procedures over
the simple staining techniques.
List and briefly describe the functions/purpose of each of the
different stains used in acid fast staining.
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INSTRUCTION: EXPLAIN THOROUGHLY THE TWO THEORIES INVOLVED IN GRAM STAINING (CELL WALL AND LIPIDS THEORIES) IN RELATION TO THE TWO STATEMENTS BELOW.
In THEORY OF CELL WALL, Gram-positive bacteria have heavy peptidoglycan, which helps decolorizer (alcohol) to dehydrate the gram-positive cell wall and traps the crystal violet complex inside the cell wall and maintains the purple color of crystal violet.Â
In THEORY OF LIPIDS, Gram-negative bacteria have high lipid amount (10-15%) in the cell wall, which makes decolorizer (alcohol) easily to remove the crystal violet complex, and then colorless cell wall is stained with safranin and appears pink/red color.
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