Session 3 lab

Flagella staining (2)

Because they are so thin, flagella typically cannot be seen under a light microscope without a specialized flagella staining technique. Flagella staining thickens the flagella by first applying mordant (generally tannic acid, but sometimes potassium alum), which coats the flagella; then the specimen is stained with pararosaniline (most commonly) or basic fuchsin

Capsule staining (4)

Certain bacteria and yeasts have a protective outer structure called a capsule. Since the presence of a capsule is directly related to a microbe's virulence (its ability to cause disease), the ability to determine whether cells in a sample have capsules is an important diagnostic tool. Capsules do not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is typically used for capsule staining. The dye stains the background but does not penetrate the capsules, which appear like halos around the borders of the cell. The specimen does not need to be heat-fixed prior to negative staining.

Gram stain procedure

It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques.

Note:

Know the structure of yeast versus hyphae drawings

What organisms are measured in milli

Worms/ Helminths and molds

Identify the 2 morphological forms of fungi(4)

Yeast and hyphae Yeast- unicellular fungi Hyphae- multicellular fungi, molds form hyphae Sporangium, makes spores that are able to make more fungi

What is the basic unit for volume

liter (L)

Types of simple stains

negative, basic and acidic stains

What organisms are measured in micro

Bacteria/ yeast and Protozoa

Acid- fast stain(3)

After staining with basic fuchsin, acid- fast bacteria resist decolonization by acid- alcohol. Non acid- fast bacteria are counter stained with methylene blue Purpose: used to distinguish acid- fast bacteria such as m tuberculosis from non-acid fast cells Outcome: acid- fast bacteria are red, non acid fast cells are blue

Gram staining gives you information about the

Bacteria - type cell wall specific

Under what circumstances are organisms best viewed for fixed, stained samples?

Dead organism or if It's dangerous organism

Positive versus negative stain(4)

Dyes are selected for staining based on the chemical properties of the dye and the specimen being observed, which determine how the dye will interact with the specimen. In most cases, it is preferable to use a positive stain, a dye that will be absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out against the background. there are scenarios in which it is advantageous to use a negative stain, which is absorbed by the background but not by the cells or organisms in the specimen. Negative staining produces an outline or silhouette of the organisms against a colorful background

steps of gram staining(4)

First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the cells a purple color. Next, Gram's iodine, a mordant, is added. A mordant is a substance used to set or stabilize stains or dyes; in this case, Gram's iodine acts like a trapping agent that complexes with the crystal violet, making the crystal violet-iodine complex clump and stay contained in thick layers of peptidoglycan in the cell walls. Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. Cells that have thick peptidoglycan layers in their cell walls are much less affected by the decolorizing agent; they generally retain the crystal violet dye and remain purple. However, the decolorizing agent more easily washes the dye out of cells with thinner peptidoglycan layers, making them again colorless. Finally, a secondary counterstain, usually safranin, is added. This stains the decolorized cells pink and is less noticeable in the cells that still contain the crystal violet dye.

Flagella stain(3)

Flagella are coated with a tannic acid or potassium alum mordant then stained using either para Rosalind or basic fuchsin Purpose: used to view and study flagella in bacteria that have them Outcome: Flagella are visible if present

Differential stains types (5)

Gram stain, acid fast stain, endospore stain Flagella stain Capsule stain

Endospores staining(7)

Gram staining alone cannot be used to visualize endospores, which appear clear when Gram-stained cells are viewed. Endospore staining uses two stains to differentiate endospores from the rest of the cell. —-The Schaeffer-Fulton method (the most commonly used endospore-staining technique) uses heat to push the primary stain ( malachite green) into the endospore. —-Washing with water decolorizes the cell, but the endospore retains the green stain. The cell is then counterstained pink with safranin . The resulting image reveals the shape and location of endospores, if they are present . The green endospores will appear either within the pink vegetative cells or as separate from the pink cells altogether. If no endospores are present, then only the pink vegetative cells will be visible

Rank the following organisms from smallest to largest (7)

HIV Staphylococcus aureus Candida albicans Trichomonas- causes trich Penicillium- where we get penicillins Pine worm- clog the intestine Tapeworm- largest isolated was 33 feet, causes anemia and malnutrition

simple versus differential staining(4)

In simple staining, a single dye is used to emphasize particular structures in the specimen. —-A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism. differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms in a differentially stained sample may appear to be different colors. ——Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining.

What measurements are the most important for microbiology

Milli, micro, nano

How do the sizes of bacteria, yeast, mold, and Protozoa viruses and proteins compare to each other? Largest to smallest

Mold, Protozoa, yeast, bacteria , viruses, proteins

Mold(6)

Multicellular •Hyphae - long, thread-like strings of cells •Mycelium- tangled mass of hyphae Reproduction •Asexual - fission or spore formation •Sexual - Spore formation

Hanging drop mount (2)

Need a coverslip, concave slide Take vasiline on edges of coverslip and put yeast or specimen in the middle and then add the concave slide

Capsule stain (3)

Negative staining with India ink or nigrosin is used to stain the background leaving a clear area of the cell and the capsule Used to distinguish cells with capsules from those without Capsules appear clear or as halos If present

Preparations and staining for other microscopes(3)

Samples for fluorescence and confocal microscopy are prepared similarly to samples for light microscopy, except that the dyes are fluorochromes. Stains are often diluted in liquid before applying to the slide. Some dyes attach to an antibody to stain specific proteins on specific types of cells ( immunofluorescence); others may attach to DNA molecules in a process called fluorescence in situ hybridization (FISH), causing cells to be stained based on whether they have a specific DNA sequence.

Preparing specimens for electron microscopy

Samples to be analyzed using a TEM must have very thin sections.

how to make a wet mount slide(3)

Slide coverslip and flame Put one small drop of water on the slide Take the S cereviseae with loop and put it into water and add the coverslip to examine under miscope

Basic stains (3)

Specific dyes: Methylene blue, crystal violet and safranin Purpose: stain negatively charged molecules and structures such as nucleic acids and proteins Outcome: positive stain

Gram stain (3)

Specific dyes: cystal violet, gram's iodine and ethanol Purpose: used to distinguish cells by cell- wall type gram negative, gram positive Outcome: gram positive cells stain purple/ violet. Gram negative stain pink

Acidic stains(3)

Specific dyes: eosin, acid fuchsin, rose bengal Purpose: stain positively charged molecules and structures, such as proteins Outcome: can be either a positive or negative stain, depending on the cell's chemistry

Negative stains

Specific types: India ink, nigrosin Purpose: stains background not specimen Outcome: dark background with light specimen

Why are unstained bacterial specimens more difficult to view than stained specimens?

Stains provide contrast between the cells and the background. Bacteria are made up of mostly water and are difficult to observe without stain because they are clear

Wet mount (5)

The simplest type of preparation is the wet mount, in which the specimen is placed on the slide in a drop of liquid. Some specimens, such as a drop of urine, are already in a liquid form and can be deposited on the slide using a dropper. Solid specimens, such as a skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid used is simply water, but often stains are added to enhance contrast . Once the liquid has been added to the slide, a coverslip is placed on top and the specimen is ready for examination under the microscope.

Preparing specimens for EM (4)

To cut cells without damage, the cells must be embedded in plastic resin and then dehydrated through a series of soaks in ethanol solutions (50%, 60%, 70%, and so on). The ethanol replaces the water in the cells, and the resin dissolves in ethanol and enters the cell, where it solidifies. Next, thin sections are cut using a specialized device called an ultramicrotome Finally, samples are fixed to fine copper wire or carbon-fiber grids and stained—not with colored dyes, but with substances like uranyl acetate or osmium tetroxide, which contain electron-dense heavy metal atoms.

Why do we use a wet mount?

To see a live organism

Yeast(5)

Unicellular •Pseudohyphae - budding cells remain temporarily connected Reproduction •Asexual - Budding •Sexual - Spore Formation

Given the potential difficultly in observing an unstained microbe, why would someone bother to view a microbe using a wet mount

Used for all live organisms. All the slides we have looked at are fixed Wet mounts let you see the organism move

Endospore stain(3)

Useds heat to stain endospores with malachite green, then cell is washed and counterstained with safranin Purpose: used to distinguish organisms with endospores from those without; used to study the endospore Outcome: appear bluish- green other structures appear pink to red

What organisms are measured in nano

Viruses

In their natural state, most of the cells and microorganisms that we observe under the microscope lack

color and contrast. This makes it difficult, if not impossible, to detect important cellular structures and their distinguishing characteristics without artificially treating specimens.

Fixation or fixed specimens (3)

fixation. The "fixing" of a sample refers to the process of attaching cells to a slide. Fixation is often achieved either by heating ( heat fixing) or chemically treating the specimen. In addition to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation.

Sample preparation for two-photon microscopy is similar to

fluorescence microscopy, except for the use of infrared dyes. Specimens for STM need to be on a very clean and atomically smooth surface. They are often mica coated with Au(111). Toluene vapor is a common fixative.

The purple, crystal-violet stained cells are referred to as(6)

gram-positive cells, while the red, safranin-dyed cells are gram-negative However, there are several important considerations in interpreting the results of a Gram stain. —First, older bacterial cells may have damage to their cell walls that causes them to appear gram-negative even if the species is gram-positive. ——Thus, it is best to use fresh bacterial cultures for Gram staining. —Second, errors such as leaving on decolorizer too long can affect the results. In some cases, most cells will appear gram-positive while a few appear gram-negative ———This suggests damage to the individual cells or that decolorizer was left on for too long; the cells should still be classified as gram-positive if they are all the same species rather than a mixed culture.

What is the basic unit for mass DOUBLE CHECK THIS

grams

Acid- fast stains(3)

is able to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls, and those that do not. Two different methods for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun technique. —-Both use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin even after a decolorizing agent (an acid-alcohol solution) is applied. A secondary counterstain, methylene blue, is then applied, which renders non-acid-fast cells blue.

If the chromophore is

is the positively charged ion, the stain is classified as a basic dye; if the negative ion is the chromophore, the stain is considered an acidic dye.

To heat- fix a sample a thin layer of the (3)

specimen is spread on the slide (called a smear), and the slide is then briefly heated over a heat source Chemical fixatives are often preferable to heat for tissue specimens. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde can denature proteins, stop biochemical reactions, and stabilize cell structures in tissue samples

In addition to fixation,(3)

staining is almost always applied to color certain features of a specimen before examining it under a light microscope. Stains, or dyes, contain salts made up of a positive ion and a negative ion. Depending on the type of dye, the positive or the negative ion may be the chromophore (the colored ion); the other, uncolored ion is called the counterion.

Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to (4)

stick to the cell walls, making them positive stains. —-Thus, commonly used basic dyes such as basic fuchsin, crystal violet, malachite green, methylene blue, and safranin typically serve as positive stains On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains. —-Commonly used acidic dyes include acid fuchsin, eosin, and rose bengal.

When samples are prepared for viewing using an SEM(4)

they must also be dehydrated using an ethanol series. However, they must be even drier than is necessary for a TEM. Critical point drying with inert liquid carbon dioxide under pressure is used to displace the water from the specimen. After drying, the specimens are sputter-coated with metal by knocking atoms off of a palladium target, with energetic particles. Sputter-coating prevents specimens from becoming charged by the SEM's electron beam.

There are two basic types of preparation used to view specimens with a light microscope

wet mounts and fixed specimens.