Micro Lab 2 essay

Staining Name: ————-
20 pts.
Objectives:
1.- Know the steps to prepare a smear of bacteria for microscopic examination.
2.- Explain the purpose of staining bacterial cells. Explain purpose of heat fixing a smear
3.- Identify the general characteristics, and morphologies of microbes that can be visualized by staining.
4.- Learn how to correctly prepare and simple stain a bacterial smear from a liquid culture, and from a colony
5.- Explain the differences between simple staining, and differential and structural stains.
6.- Describe how the Gram stain works and summarize the differences in cell wall structure of gram positive and gram-negative cells. List the Gram stain reagents, steps, and purpose of each. Recognize some common mistakes that can affect a Gram stain.
7.- Describe the purpose, reagents, and procedure of the acid-fast stain. Know the genus of medically important acid-fast organisms. Recognize some common mistakes that can affect an Acid-Fast Stain
8.- Describe endospores and the differences between endospore and vegetative cells. Name bacteria genera that are endospore formers.
9.- Describe the purpose, reagents, and procedure of the endospore stain. Recognize some common mistakes that can affect an endospore stain.
10.- Describe the structure and function of bacterial capsules. Describe a negative staining technique. Describe the purpose, reagents, and procedure of the capsule stain. Recognize some common mistakes that can affect a capsule stain.1.- Staining: Smear Preaparation (3.0 pts.)
A smear is a sample of bacteria taken from a culture and spread onto a clean microscope slide
1.- (1 pt.) Use the space below to load the picture of an unstained vs. stained sample from the virtual lab exercise. Use the pictures to explain the purpose of staining.2.- (1 pt.) Staining problems: Heat fixing
What happens if the smear is not heat fixed long enough? Or heat fixed for too long?What happens is the smear is prepared using an excessive amount of sample?What happens if the smear is prepared using not enough sample?Bacterial smears can be prepared from broth or agar media, or from clinical specimens.
3.- (1 pt.) When preparing a smear from a Petry plate, the sample of bacteria is picked from an isolated colony. What is an isolated colony?Why is it important to pick the sample from one isolated colony?2.- Bacterial Staining: Introduction (1 pts.)
1.- (1 pt.) Match the reagents used in a differential with their purpose:
———-Decolorizer A.- second dye applied during a differential stain.
———-Primary stain B.- removes the first dye from a bacterial cell
———-Countersatin C.- first dye applied during a differential stain3.- Bacterial Staining: Gram Stain (5 pts.)
The gram stain is the most important stain in microbiology. It helps to identify and characterize bacteria which leads to choosing an appropriate treatment method.
1.- (1 pt.) Give two differences between Gram positive, and Gram-negative cell walls.2.- (1 pt.) List the reagents used in a gram stain by writing their names next to the functions listed.—————- Primary stain —————-mordant —————decolorizer
—————–counter stain
What is the function of the mordant?3.- (1 pt.) Explain why gram positives retain the primary stain -mordant complex during decolorization.Describe the color of the gram positive and gram-negative cells after a gram stain, and the reasons for the colors.4.- (1pt.) Describe the gram reaction and shape of all the bacteria observed under the microscope.
**Include a picture of the specimen viewed under the microscope.6.- (1 pt.) Problems with gram staining:
What happens to a gram positive if it is over-decolorized?What happens to a gram negative if it is under-decolorized?What happens if the counterstain is omitted in a mixed culture?What if you forget the crystal violet step?4.-Bacterial Staining: Acid Fast Staining (4.0 pts.)
1.- (1 pt.) What is the purpose of the acid-fast stain?Name a genus and species of a medically important acid-fast bacteriaName the lipid that makes the wax in the acid-fast cell wall2.- (1 pt.) List the dyes and reagents used during an acid-fast stain, and explain their purpose3.- (1pt.) Describe the acid -fast sample. Describe the shape and color of the acid fast and non-acid fast cells. **Include a picture of the specimen viewed under the microscope.4.- (1 pt.) Problems the with acid-fast stain:
If the carbolfuchsin is omitted from the procedure what color are the acid-fast cells?What would happen to the non-acid fast cells if the counterstain is omitted?5- Bacterial Staining: Endospore Staining (4 pts.)
1.- (1 pt.) Distinguish vegetative cells from endosporesName two genera of endospore former bacteria2.- (1 pt.) List the reagents used during the endospore stain and their purpose.3.- (1pt.) Describe the result of the endospore stain. **Include a picture of the specimen viewed under the microscope.5.- (0.5 pts.) An endospore stain is performed using a non-endospore forming bacteria. Describe what the slide will look like when viewed under the microscope.6.- ( 0.5 pt.) Problems with the endospore stain.
What happens if the counterstain is omitted?
What happens if the malachite green is omitted?6.- Bacterial Staining: Capsule Stain (3.0 pts.)
1.-(0.5 pt.) What is the purpose of a capsule stain?What are the functions of a capsule?Capsules cannot be stained because they are stable structures that do not create chemical bonds with stains. To detect the presence of capsules, a negative staining technique is used that stains the background and the cell inside the capsule, leaving the capsule unstained.2.- (1 pt.) What type of stain, acidic or basic, is used to stain the background during a capsule stain?
What type of stain is used to stain, acidic or basic, the cell?NOTE: The virtual lab incorrectly calls slime layers capsules. Slime layers and capsules are glycocalyx structures. However, they are not the same. Refer to Chapter 3 power points for the differences in structure, and function of these two types of layers.3.- (1pt.) Describe what the sample looks like in the microscope. **Include a picture of the specimen viewed under the microscope.
What is the shape of the bacteria? Describe (or label) the capsule.4.- (0.5 pt.) Problems with the capsule stain.
What happens if the basic stain is omitted?What happens if the slide is heat fixed?7.- Xtra points Review (5 pts.)1.- (3 pts.) Match the staining procedure with its purpose
Quick method to determine bacteria morphology and arrangements –—————-
Which stain does not require heat fixation –——–
A stain that can differentiate between cells that have a thin and a thick layer of peptidoglycan in their cell wall —————–
A stain that identifies cells with mycolic acid in their cell wall ——————
A stain that uses heat to penetrate tough layers around a bacterial structure. ———
Staining procedures that uses safranin as the counterstain ———————
Staining procedure that uses methylene blue as the counter stain ——————
Staining procedure that uses a mordant —————–
Staining procedure that uses negative staining technique ——–
Staining technique that uses water to remove the primary stain ———2xtra points: Challenge question.
You are a microbiology lab technician who has received the following clinical specimens (listed below). Determine the best staining procedure to identify the bacteria causing the infection in the patient.
Here are the organisms involved in the infections. You need to match the organism causing the infection with the clinical specimen (A-D), and determine the staining procedure that would help identify the organism
Klebsiella pneumoniae Staphylococcus aureus Clostridium perfringens Mycobacterium tuberculosis
Clinical specimens received:
A.- Sputum sample from a patient with a persistent cough, mild fever, and blood-tinged sputum specimen.
B.- A specimen from an infected wound
C.- A respiratory exudate from a patient with pneumonia.
D.- A tissue specimen from a patient with gas gangrene
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