SMEAR PREPARATION - Microbiology Lab

SIMPLE STAINING PROCEDURE (after you create your smear)

1. Add methylene blue dye to slide smear. Wait 1 minute. 2. Rinse slide with distilled water. 3. Move slide to blotting paper and fold it over to remove excess stain and liquid from sample. 4. Move slide to microscope to observe :)

Name the STEPS for PREPARING A BACTERIAL SMEAR FROM A LIQUID CULTURE:

1. Label slide with marker. 2. Sterilize loop by placing into flame of Bunsen burner. 3. Pick up culture tube. Remove cap. Briefly heat mouth of tube in Bunsen Burner flame to reduce contamination. 4. Insert loop into tube to acquire bacterial sample. 5. Briefly heat mouth of tube. Replace cap. Return tube to rack. 6. Smear bacteria across center of slide. 7. Sterilize loop using Bunsen Burner. Return loop to holder. Allow bacterial smear to air dry for 5 minutes. 8. Using clothespin to hold slide, perform heat fixation of sample by passing through the Bunsen Burner for 3-5 seconds. Return slide to staining tray.

Name the STEPS for PREPARING A BACTERIAL SMEAR FROM A SOLID MEDIA.

1. Label slide with marker. 2. Sterilize loop by placing into flame of Bunsen burner. 3. Use loop to retrieve a drop of water from beaker. Add water drop to center of slide. 4. Reheat loop in Bunsen burner flame to reduce possible contamination. Acquire bacterial sample from plate. 5. Use loop to mix bacteria into water drop on slide. Spread sample material to make a thin smear. 6. Sterilize loop using Bunsen burner. Return loop to holder. Allow bacterial smear to air dry for 5 minutes. 7. Using clothespin to hold slide, perform the heat fixation of sample by passing through the Bunsen Burner for 3-5 seconds. Return slide to staining tray.

Methylene blue

A blue dye used to visualize cells in bacteriological and biological staining (C16H18CIN3S).

Liquid broth culture

A liquid nutrient medium used for the propagation of microorganisms.

Aseptic technique

A method of handling microbial cultures, patient specimens, and other sources of microbes in a way that reduces contamination and prevents infection of the handler or others who may be exposed.

Where can a bacterial smear be stored?

A properly performed and fixed bacterial smear can be stored in a dry place for months before further use.

Based on what you learned in this simulation, for which of the following samples would you skip the heat fixation step???

A sample of pond water, looking for living bacterial activity such as observing for motility

What is a smear prep?

A smear prep is a technique used to produce a sample of microorganisms, typically for microscopy or staining. While there are many variations of the smear prep technique, depending on the final desired use of the sample, the overall key steps are the same for a majority of smear prep protocols.

Agar media

A solid nutrient medium used for the propagation of microorganisms.

Inoculating loop

A sterilizable tool made of wire, used to transfer microorganisms.

Staining

A technique that utilizes dye(s) to add color for visualizing and distinguishing cells on a smear by increasing contrast.

Smear

An isolated and prepared sample of microorganisms for use in microscopy and staining.

Bacterial smears are most often created from _________ or ____________.

Bacterial smears are most often created from either 1) bacteria in liquid broth culture or 2) bacteria on agar media

Learning how smear preparation works is critical to good staining and microscopy outcomes.

Correct smear preparation provides scientists with significant information about the sample, such as: = bacterial cellular arrangement = bacterial cellular morphology = bacterial structures

Bacterial cellular arrangement

Description of overarching patterns and organization of bacterial cells seen across a microscopic field. Examples include descriptors such as "clusters" or "in pairs."

Bacterial cellular morphology

Description of shapes and structure of bacterial cells seen across a microscopic field. Examples include descriptors such as cocci or bacilli.

Name the steps in preparing a smear!

From Solid Media: 1. Two loopfuls of water are placed in center of slide. 2. A very small amount of a solid culture is dispersed with inoculating needle in water over middle 1/3 slide. 3. the smear is allowed to dry at room temperature. 4. Slide is passed through flame several times top heat-kill and fix organisms to slide. From Liquid Media: 1. Two loopfuls of liquid containing organisms are placed in center of slide. 2. Culture is dispersed over middle 1/3 of slide. 3. the smear is allowed to dry at room temperature. 4. Slide is passed through flame several times top heat-kill and fix organisms to slide.

In this simulation, the heat fixation was performed for you. Because each sample is unique, there is no "set" time for completing heat fixation. You should wait until the smear is totally air dry to prevent boiling your sample off. Then, gentle, gradual heating is needed to both fix to the slide and preserve the sample integrity.

In a real lab experiment, students are often told to perform the fixation step by waving the slide back and forth through the flame several times and then to check the temperature of the slide by quickly touching the underside of the slide to the back of your hand. In general, if the slide is too hot to touch, the heat fixation has gone on too long and the cells may be damaged or lysed. Consult your lab instructor or lab protocols at your school for more information on this procedure.

Fields of study where smear preparation and staining are necessary tools include:

Microbiology: Microbiology is the study of microorganisms. Cytology: Cytology is the study of cells. Histology: Histology is the study of tissues. Pathology: Pathology is the study of disease.

In this simulation, you will practice the preparation of the two major types of bacterial smears - simple and differential.

Most bacteria are too small to be viewed by the naked eye; therefore, they must be visualized using some type of microscope. Additionally, a colored chemical dye, known as a stain, is often added to provide additional contrast to help visualize the microbial cells.

Microscopy

Science that studies structure, magnification, lenses, and techniques related to use of a microscope.

The addition of methylene blue helps: Now that you have seen the difference in the smear preparation after simple methylene blue staining, which of the following is correct regarding your smear now?

The addition of methylene blue helps: = visualize the shape & arrangements of bacterial cells that were difficult to discern without stain. = show the intact appearance and localization of the cells, compared with no staining. = makes visualizing some bacterial cell characteristics more apparent, compared to no staining.

What do the basics of a smear preparation involve?

The basics of a smear preparation involve 5 things: = Growing a bacterial culture in liquid nutrient broth or on an agar plate = Acquiring a portion of the sample for smear creation using aseptic technique = Applying the sample to a slide to distribute the organisms = Heat fixation of the smear to adhere the sample to the slide = Evaluation of the quality of the smear right away using a simple stain called methylene blue

Heat fixation

The process of applying gentle heat to a sample. Fixing a specimen preserves various cellular components in a natural state with minimal distortion, while simultaneously killing the specimen and securing it to the slide.