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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

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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

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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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  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

.

How to perform aseptic technique when inoculating from a test tube




Figure:


  1. Label the tube to be inoculated with the name of the organism and your initials.
  2. Place the tubes in the palm of your hand, secure with your thumb, and separate to form a V.
  3. Flame the needle or loop until the entire end is red.
  4. With the sterile loop or needle in hand, uncap the tubes.
  5. Flame the necks of the tubes by rapidly passing them through the flame once.
  6. Slant-to-broth transfer: Dislodge inoculum by slight agitation. Broth-to-slant transfer: Following insertion to base of slant withdraw the loop in a zigzag motion. Slant-to-agar deep transfer: Insert the needle to the bottom of the tube and withdraw along the line of insertion.
  7. Flame the necks of the tubes by rapidly passing them through the flame.
  8. Recap the tubes.
  9. Reflame the loop or needle.



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

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How to perform aseptic technique when inoculating from an agar plate




Figure:


  1. Flame the straight needle until the entire wire is red.
  2. After inoculating a discrete colony on the agar streak plate, touch the straight needle to the surface of the selected colony.
  3. Uncap the agar slant and pass the neck of the tube rapidly over the Bunsen burner flame.
  4. Inoculate the slant by drawing the needle upward in a zigzag motion along the surface of the agar. Do not dig into the agar.
  5. Flame the neck of the tube and recap.
  6. Flame the inoculating needle.

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

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  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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by PNG: Wikipedysta:Reytansvg: Marek M (talk) 11:44, 3 May 2011 (UTC), Public domain, via Wikimedia Commons is licensed under

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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


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



Streaking a plate using an inoculation loop


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by Lucas R, CC BY-SA 4.0

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Campylobacter jejuni

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


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by Kubo Michal is licensed under

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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)


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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 plate method: The sample is poured onto solid agar and then spread using a sterile spreader. This process is repeated for each serial dilution prepared.


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The

pour-plate method

also requires inoculation from a serial dilution of the mixed culture by means of a loop or a pipette. The diluted inoculum is then added to a molten agar medium in a Petri dish, mixed, and allowed to solidify. The serial-dilution agar plate technique is used to quantitate viable cells. This method involves serial dilution of a bacterial suspension in sterile water banks, which serves as a diluent of known volume.

Once diluted, the suspensions are placed on suitable nutrient media. The pour plate technique uses molten agar cooled to 45 ºC and poured into a Petri dish containing a specific amount of the diluted sample. Following addition of the molten-then-cooled agar, the cover is replaced, and the plate is rotated in a circular motion to achieve uniform distribution of microorganisms. This procedure is repeated for all dilutions to be plated, incubated overnight, and counted.

Plates suitable for counting must contain no fewer than 30 or no more than 300 colonies. The total count of the suspension is obtained by multiplying the number of cells per plate by the dilution factor, which is the reciprocal of the dilution.

Some advantages of the serial dilution agar plate technique are that only viable cells can be counted and the technique allows isolation of discrete colonies that can be subcultured into pure cultures.

Some disadvantages of the technique are (1) overnight incubation is necessary before colonies develop on the agar surface; (2) it is necessary to use more glassware than in the streak-plate or spread-plate methods, and (3) the need for greater manipulation may result in erroneous counts due to errors in dilution or plating.



Pour plate method: the sample is mixed in liquid warm agar (45–50 ºC) poured into a sterile Petri dish and further mixed by swirling. This process is repeated for each serial dilution prepared.


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When grown on a variety of media, microorganisms will exhibit differences in the macroscopic appearance of their growth. These differences, called

cultural characteristics

, are used as the basis for separating microorganisms into taxonomic groups. The cultural characteristics for all known microorganisms are contained in Bergey’s Manual of Systematic Bacteriology. They are determined by culturing the organisms on nutrient agar plates, on nutrient agar slants, and in nutrient broth.


Agar plates

are sterile Petri plates that are aseptically filled with a melted sterile agar medium and allowed to solidify. Plates are much less confining than slants and are commonly used in the culturing, separating, and counting of microorganisms. Nutrient agar plates demonstrate well-isolated colonies and are evaluated for size, pigmentation, form, margin, and elevation.

  • The size of the colonies can be pinpoint, small, moderate, or large.
  • The pigmentation is the color of the colony. Colonies may be pigmented (purple, yellow, red, etc.) or nonpigmented (cream, tan, or white).
  • The form of the colonies constitutes the shape of the colony.

    • The form of the colony can be circular, irregular, or rhizoid.
  • The margin is the appearance of the outer edge of the colony.

    • The margin can be entire (sharply defined; even), lobate (indentations), undulate (wavy), serrate (tooth-like), or filamentous (thread-like; spreading).
  • The elevation is the degree to which colony growth is raised on the agar surface.

    • The elevation of the colony can be flat (elevated; not discernible), raised (slightly elevated), convex (dome-shaped elevation), or umbondate (raised, with elevated convex central region).



Colony morphologies


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Agar slant tubes

are tubes containing a nutrient medium plus a solidifying agent such as agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface. Nutrient agar slants have a single straight line of inoculation on the surface and are evaluated in the following manner:

  1. Abundance of growth: The amount of growth is designated as none, slight, moderate, or large.
  2. Pigmentation: Chromogenic microorganisms may produce intracellular pigments that are responsible for the coloration of the organisms as seen in surface colonies. Other organisms produce extracellular soluble pigments that are excreted into the medium and that also produce a color. Most organisms, however, are nonchromogenic and will appear white to gray.
  3. Optical characteristics: May be evaluated based on the amount of light transmitted through the growth. Described as opaque (no light transmission), translucent (partial transmission), or transparent (full transmission).
  4. Form: The appearance of the single-line streak of growth on the agar is designated as filiform (continuous; smooth edges), echinulate (continuous; irregular edges), beaded (nonconfluent), effuse (thin; spreading), arborescent (tree-like), or rhizoid (root-like).



Patterns of growth on agar slants


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Nutrient broth tubes

are tubes containing a liquid medium. A typical nutrient containing broth medium, such as trypticase soy broth, contains substrates for microbial growth. After incubation, growth (development of many cells from a few cells) may be observed.

Nutrient broth cultures are evaluated as to the distribution and appearance of growth: uniform fine turbidity (finely dispersed cloudiness throughout), flocculent (flaky aggregates dispersed throughout), pellicle (thick, pad-like growth on surface), and sediment (concentration of growth at the bottom of broth culture may be granular, flaky, or flocculent).



Microbial growth in broth media


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Assessing Your Learning


Warning:

You are expected to submit your own, individual work. Using work completed by anyone other than yourself is plagiarism. This includes resources found on Internet sites. Posting assessments on an unauthorized website, soliciting assessment answers or the acquisition of assessments, assessment answers, and other academic material is cheating. Cheating and/or plagiarism will result in a failing grade for the course.

Assignments


Submit

LAB 3


.

Important information:

Copy and paste the list of Laboratory Exercise Questions into a Word document. Compose answers to these questions in the Word document and save the file as a backup copy in the event that a technical problem is encountered while attempting to submit the assignment. Make sure to run a spell check.

You will be submitting your answers to the lab assignment in two parts. The first part of the lab assignment consists of the laboratory exercise questions. The second part of the lab assignment is the application question. The first textbox on the submission page corresponds to the first part of the lab. Be sure to paste the laboratory exercise questions, with your answers, into this textbox. The second textbox on the submission page will be for your response to the application question.


Laboratory Exercise Questions




~~1. According to the video, what are 3 reasons aseptic technique is necessary? (3 points)


~~2. Why is it necessary to flame the inoculation loop as part of aseptic technique? Is this procedure considered disinfection or sterilization of the loop? Explain your answer. (2 points)

~~3. Can a pure culture be prepared directly from a mixed-broth or a mixed-slant culture? Explain. (2 points)

~~4. As demonstrated in the video, why do you pass the mouth of the tube through the flame? What is the correct angle? (2 points)

~~5. How do you know that you have used aseptic technique correctly and have achieved a pure culture? (2 points)

~~6. What are some advantages and disadvantages of the serial dilution agar plate technique? (2 points)

~~7. Describe at least 2 different cultural characteristics identified when you use the following. (6 points)

a. Agar slants:

b. Nutrient broth:

c. Agar plates:

~~8. Using your own wording, distinguish between the following. (2 points)

a. Antisepsis versus disinfection:

b. Cleaning versus sterilizing:

~~9. What is an advantage of an agar plate over a slant tube? (1 point)

~~10. Describe how discrete colonies can appear from a mixed culture using the streak-plate subculture process. (2 points)


Application Question

~~11. How might the information gained from this lab pertaining to aseptic technique be useful to you as a healthcare professional? (6 points)


Key components of critical thinking and application include the following:

  1. Demonstrates application and comprehension of the scientific principles. (40%)
  2. Displays competence in applying scientific knowledge to your professional life. Relevant content is supported by facts, data, and detailed examples. (40%)
  3. The application paragraph is organized and structured. (10%)
  4. Use of accurate scientific terminology. (10%)
Critical Thinking and Application of Information 0% 1-59% 60-89% 90-100%

Is your application a detailed description of how the lab content is relevant to your life?
Application did not adequately demonstrate application or comprehension of the scientific principles. Did not include detailed examples, facts or data. Or the application was not included. A few areas of the application demonstrated some application and comprehension of the scientific principles by applying the knowledge to the student’s personal and professional life, but lacked detailed examples to support the content provided. Application demonstrated some organization and structure within the paragraph. Most areas of the application demonstrated evidence of critical thinking and comprehension of the scientific principles. Displayed good competence and the relevant content was supported with good use of examples that apply the concepts and describe how the information will be relevant and useful to the student’s personal or professional life. The application paragraph is primarily presented in an organized and structured manner. Application included a complete and detailed description of how the concepts are relevant and useful or applicable to the student’s personal or professional life. The application includes detailed examples and reveals insight into the scientific principles. The application paragraph maintains a strong sense of purpose and organization throughout.

Summarizing Your Learning


Have You Met The Objectives For This Lab?

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