Lab 3

Aseptic Technique

Introduction: Connecting Your Learning

Microorganisms are always present in the air and on laboratory surfaces, benches, and equipment. They can serve as a source of external contamination and interfere with experimental results unless proper techniques are used to prevent contamination. The technique that is most often used to prevent the contamination of culture is called the aseptic technique. This technique is important for the growth, isolation, and identification of microorganisms in the laboratory.

Readings, Resources, and Assignments

Multimedia Resources	Aseptic Technique Video
Required Assignments	Lab 3 Assignment

Focusing Your Learning

Course Competency

1. Utilize aseptic technique for safe handling of microbes.

As you work through this lab, consider the objectives below.

Lab Objectives

By the end of this lab, you should be able to:

1. Identify techniques for aseptic handling, transfer, and disposal of microbial organisms in the lab.
2. Define how to maintain cultures, emphasizing transfer and isolation of microorganisms.

Background Information

Please watch and take notes on this aseptic technique video: Aseptic Technique Video

Aseptic technique
In nature, most microbes are found growing in environments that contain many different organisms. Unfortunately, these mixed cultures are of little use in studying microorganisms because of the difficulty in determining which organisms are responsible for any observed activity. In order to grow and identify the microorganisms, the microorganisms must be isolated from one another in the specimen.

This process is done by transferring the microorganisms from one vessel to another or from culture to various media for maintenance and study. Such a transfer is called subculturing and must be carried out under sterile conditions to prevent possible contamination. The purpose of subculturing is to obtain separate cultures, each containing only one species of microorganism. A culture containing a single unadulterated species of cells is called a pure culture. Pure cultures contain only one type of organism and are suitable for the study of their cultural, morphological, and biochemical properties. This is of extreme importance in preparing and maintaining cultures as well as in microbiological test procedures. To isolate and study microorganisms in pure culture, the microbiologist requires basic techniques such as aseptic technique.

Subculturing enables one to go from a mixed culture plate that contains many different colonies to a pure culture containing just one type of microbe.

This agar plate contains mixed cultures from a swab performed at a hospital on patient elevator buttons and escalator rails.

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by Jnims, CC BY-SA 3.0 , via Wikimedia Commons is licensed under CC BY 4.0.

A blood agar plate containing a of pure culture Staphylococcus

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by Bill Branson – (Edited by Fir0002)(Edited by Drhx), Public domain, via Wikimedia Commons is licensed under CC BY 4.0.

Aseptic technique encompasses many different processes. Aseptic technique includes the use of gloves to prevent contamination from people, the use of heat to kill microorganisms on transfer equipment such as needles or loops, and transfer techniques to isolate microorganisms on culture media.

The following table summarizes basic vocabulary concerning the aseptic discussion.

Term	Definition
Antisepsis	Refers to the killing or removal of microbes on living tissues. Sepsis is bacterial contamination.
Clean	“Clean” has a very restricted meaning in microbiology. Generally it means the removal of dirt or debris superficially. It does not kill or remove all microbes.
Disinfection	Refers to the killing of microbes on inanimate objects or materials (non-endospore-forming).
Germicide/Biocide	A chemical agent that demonstrates killing power against various microbes. -cide = means to KILL (excluding endospores).
Pasteurization	A heating process that reduces the number of spoilage germs and eliminates pathogens in milk and other heat sensitive foods.
Sanitization	Usually used by the food industry. Reduces microbes on eating utensils to safe, acceptable levels for public health. Generally reduces microbial counts.
Static	Processes or chemical agents that inhibit bacterial growth but do not necessarily kill microbes.
Sterilization	Kills or removes all forms of life, including bacterial endospores.

The microbial growth on this plate is the result of contamination. Multiple colonies exhibiting various different cultural characteristics are present.

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by Garnhami, CC BY-SA 4.0 , via Wikimedia Commons is licensed under CC BY 4.0.

Described below are essential steps that one must follow for aseptic transfer of microorganisms.

1. An inoculating needle or loop must always be sterilized by holding it in the hottest portion of the Bunsen burner flame, the inner blue cone, until the entire wire becomes red-hot. An incinerator can be used instead of a Bunsen burner. Then, the upper portion of the handle is rapidly passed through the flame. Once flamed, the loop is never put down but is held in the hand and allowed to cool for 10-20 sec. The stock culture tubes and the tube to be inoculated are held in the palm of the other hand and secured with the thumb. The two tubes are then separated to form a V in the hand.

Preparing to flame a loop

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by Samuel Larson, CC BY 4.0 , via Wikimedia Commons is licensed under CC BY 4.0.

1. The tubes are uncapped by grasping the first cap with the little finger, the second cap with the next finger, and lifting the closures upward. Once removed, these caps must be kept in the hand that holds the sterile inoculating loop or needle, thus the inner aspects of the cap point away from the palm of the hand. They must never be placed on the laboratory bench because doing so would compromise the sterile procedure. Following removal of the closures, the necks of the tubes are briefly passed through the flame and the sterile transfer instrument is further cooled by touching it to the sterile inside wall of the culture tube before removing a small sample of inoculum. The necks of the tubes are flamed to prevent contamination of the tube from the outside environment, to prevent the sterile needle from becoming contaminated if it touches the neck of the tube, and to prevent the bacteria inside the tube from leaving the tube.
2. Depending on the culture medium, a loop or needle is used for removal of the inoculum. Loops are commonly used to obtain a sample from a broth culture. Either instrument can be used to obtain the inoculum from an agar slant culture by carefully touching the surface of the solid medium in an area exhibiting growth so as not to gouge into the agar. A straight needle is always used when transferring microorganisms to an agar deep tube from both solid and liquid cultures.
3. The cell-laden loop or needle is inserted into the subculture tube. In the case of a broth medium, the loop or needle is shaken slightly to dislodge organisms; with an agar slant medium, it is drawn lightly over the hardened surface in a straight or zigzag line. For inoculation of an agar deep tube, a straight needle is inserted to the bottom of the tube in a straight line and rapidly withdrawn along the line of insertion. This is a stab inoculation.
4. Following inoculation, the instrument is removed, the necks of the tubes are reflamed, and the caps are replaced on the same tube from which they were removed.
5. The needle or loop is again flamed to destroy remaining organisms.

The following are examples of how to perform aseptic technique to transfer microbes:

Creative Commons by Microbiology: A Laboratory Manual 6th ed. is licensed under CC BY 4.0.

Creative Commons by Microbiology: A Laboratory Manual 6th ed. is licensed under CC BY 4.0.

Using a subculturing process, microorganisms in the specimen are repeatedly transferred from culture to culture until colonies appear that are separate from one another. Colonies are individual, macroscopically visible masses of microbial growth on a solid medium surface, each representing the multiplication of a single organism. Once the discrete colonies are obtained, an aseptic transfer is made onto nutrient agar slants for the isolation of pure cultures.

The techniques commonly used for isolation of discrete colonies initially require that the number of organisms in the inocolum be reduced. The decrease in the population ensures that, following inoculation, individual cells will be sufficiently far apart on the surface of the agar medium to cause a separation of the different species present. The following are subculturing techniques that can be used to accomplish this necessary dilution and isolation of colonies.

The streak-plate method is a rapid qualitative isolation method. It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate. In the streak-plate method, a loop is used to streak the mixed sample many times over the surface of a solid culture medium in a Petri plate. The process of streaking the loop repeatedly over the agar surface causes the bacteria to fall off the loop one by one and to be distributed over the agar surface, where each cell develops into a colony. Although many types of procedures are performed, the four-way or quadrant streak will be described.

Flaming a loop: The loop is sterilized by holding it in the hottest portion of the Bunsen burner flame, the inner blue cone until the entire wire becomes red-hot. Then it must be cooled 10-20 seconds before it can be used.

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by CSIRO, CC BY 3.0 , via Wikimedia Commons is licensed under CC BY 4.0.

1. Using a flamed and cooled loop, place a loopful of culture on the agar surface. Flame and cool the loop, then drag it rapidly several times across the surface of area.

   To avoid digging into the agar as one streaks the loop over the top of the agar, one must keep the loop parallel to the agar surface.

Step 1 to streak a plate

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1. Reflame and cool the loop and turn the Petri dish 90º. Then touch the loop to a corner of the culture in area 1, and drag it several times across the agar in area.

   The loop should never enter area 1 again.

Step 2 to streak a plate

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1. Reflame the loop and cool the loop, and again turn the dish 90º. Streak area 3 in the same manner as area 2.

Step 3 to streak a plate

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1. Without reflaming the loop, again turn the dish 90º and then drag the culture from a corner of area 3 across area 4, using a wider streak. Do not let the loop touch any of the previously streaked areas. The flaming of the loop at the points indicated is to affect the dilution of the culture so that fewer organisms are streaked in each area, resulting in the final desired separation.

Step 4 to streak a plate

Creative Commons https://commons.wikimedia.org/wiki/File:Streak_pla… by PNG: Wikipedysta:Reytansvg: Marek M (talk) 11:44, 3 May 2011 (UTC), Public domain, via Wikimedia Commons is licensed under CC BY 4.0.

The following pictures are examples of the streak-plate method:

Streaking a plate using an inoculation loop

Creative Commons https://commons.wikimedia.org/wiki/File:Utstryksme… by Lucas R, CC BY-SA 4.0 , via Wikimedia Commons is licensed under CC BY 4.0.

Campylobacter jejuni colonies isolated on blood-free, charcoal based selective medium (CSM).

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by Kubo Michal is licensed under CC BY 4.0.

The spread-plate technique requires a previously diluted mixture of microorganisms to be used.

Serial dilution dilutes a fixed volume of cells mixed with dilution solution using the previous dilution as an inoculum. The result is dilution by an exponentially growing factor. (modification of work by “Leberechtc”/Wikimedia Commons)

Creative Commons https://openstax.org/books/microbiology/pages/9-1-… is licensed under CC BY 4.0.

To make a spread plate, the cells are evenly spread over the surface of a solid agar medium using a sterile, L-shaped bent rod while the Petri dish is rotated. (This can be accomplished while the plate is spun on a lazy Susan-type turntable). The step-by-step procedure for this technique is as follows:

1. Place the bent glass rod into the beaker and add a sufficient amount of 95% ethyl alcohol to cover the lower bent portion.
2. With a sterile loop, place a loopful of culture in the center of the nutrient agar plate (that has been placed on the turntable if using the turntable). Alternatively, pour or pipette sample onto the agar. Replace the cover.
3. Remove the glass rod from the beaker, and pass it through the Bunsen burner flame with the bent portion of the rod pointing downward to prevent the burning alcohol from running down one’s arm. Allow the alcohol to burn off the rod completely. Cool the rod for 10-20 sec.
4. Remove the Petri dish cover and if using the turntable, spin the turntable.
5. Lightly touch the sterile bent rod to the surface of the agar and move it back and forth to spread the culture evenly over the agar surface.
6. Replace the cover. Immerse the rod in alcohol and reflame.

Spread plat