Micro Lab (1) Dr F, Talaro

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microbiology lab, Talaro, Foundations in Microbiology

in·oc·u·late

To introduce a serum, vaccine, or antigenic substance into (the body of a person or animal), especially to produce or boost immunity to a specific disease.
2. To communicate a disease to (a living organism) by transferring its causative agent into the organism.
3. To implant microorganisms or infectious material into (a culture medium).

labeling culture

Organize all sterile media at the start of each experiement with non-water soluable glassware markers and;or self-stick labels prior to their inoculation.name of the test organism, name of the medium, the dilution of the sample, your name or initials and the date. place labelingbelow the cap or on the bottom of Petri dish.

aseptic transfer

A minute amount of inoculum if required. If an agar culture is used, touch only a single area of growth with the incocuation wire to obtain the inoculum. Never drag the loop or needle over the entire surface, and take care not to dig into the solid medium.

Pipettes

use sterile, and place in white tub on bench when time to discard.

incubation procedure

Opptmum growth occurs at 37C, over 18 to 24 hours. Place culture tubes in a rac for incubation. Petri dishes may be stacked, however, they must alwaus be incubated in an inverted position (top down) to prevent water condensation from dropping onto the the surface of the culture medium. (pg 4 lab book).

Recording observations and results

Complete all reading prior to the experiment. Meticulously record all the ovserved data in the Lab Report of each experiement. Experiements require drawings to illustrate microbial morphology to depict shapes, arrangements, and cellular structures enlarged to 5 to 10 times their actural size.

Termination of Lab sessions

Return all equipment, place all tubes and Petri dishes in designated colllection area, place contaminates in biohazard, wipe down lab bench with disinfectant, wash hands.

Pure culture

A culture containing a single unadulterated species of cells.Microbiologists require basic lab apparatus and the application of specific technique as illustrated in figure 1.

Media

Survival and continued growth of microorganisms depend on an adequate supply of nutrients and a favorable growth environment. For survival, most microbes must use soluble low-molecular-weight substances that are frequently derived from the enzymatic degradation of complex nutrients. A solution containing the nutriens is a culture medium.

culture medium

microorganisms depend on an adequate supply of nutrients and a favorable growth environment--all culture media are liquid, semisolid, or solid. A liquid medium lacks a solidifying agent and is called a broth mediium.
A broth medium supplemented with a solidifying agent called agar results in a solid or semisolid medium.

Agar

An extract of seaweed, a carbohydrate composed mainly of galactose, and is without nutriional value. Agar serves as an excellent solidigying agent because it liquefies at 100°C and solidifies at 40°C .

Semisolid medium

A concentration of less than 1% agar results in a semisolid medium. A solid medium has the advantage that it presents a hardened surface in which microorganisms can be grown using specialized techniques for the isolation of discete colonies.

colony

is a cluster of cells that originates from the mutiplication of a single cell and represents the growth of a single species of microbes--such a defined and well -isolated colony is a pure culture. Also, while in in the liquefied state, solid media can be placed in test tubes, which are then allowed to cool and harden in a slanted position, producing agar slants.

agar slants

while in in the liquefied state, solid media can be placed in test tubes, which are then allowed to cool and harden in a slanted position, producing agar slants

agar deep

while in in the liquefied state, solid media can be placed in test tubes, which are then allowed to cool and harden in an upright position, producing agar slants--used primarily for the study of gaseous requirements of microorganisms. May be liquefied in a boiling water bath and poured into Petri dishes, producing agar plates

agar plate

liquefied in a boiling water bath and poured into Petri dishes, producing agar plates, which provide large surface areas for the isolation and study of microes. see figure (2). In addtional to nutritional needs, the environmental factors must also be regulated, including proper pH, temperture, gaseous requirements, and osmotic pressure.

Aseptic

the proces of rendering a medium or material free of all forms of life.To acheive sterility, it is mandatory that you use sterile equipment an aseptic techniques. figure (3)

test tubes and Petri dishes

suitable nutrient medium in the form of broth or agar, while only a solid medium is used in Petri dishes. Sterile environment is maintained is culture tubes by various closures. First, was cotton plug, was deveoped by Schroder and von Dusch in the ninetenth century. Most use sleevelike caps (Morton closures) made of metal, such as stainless steel, or heat-resistant plastics.Peri dishes are incubated in an inveted position to prevent condensation. Built-in ridges on tube closures and Petri dishes provide small gaps necessary for the exchange of air. figure (4) cultural vessels.

transfer instruments

microbes must be transferred from one vessel to another or from stock cultures to various media for maintenance and study called subculturing and must be carried out aseptically. Wire loops ad needlesare meade frominert metals such as Nichrome or platinum and are inserted into metal shafts that serve as handles--easily sterlized by inceneration in the blue (hottest) portion of the Bunsen burner flame. Pipettes have similar function as straws, figure (5)

cultivation chambers

Prime requirement that microbes grow at their optimum temperature. Incubators are used, most are dry heat. Moisture is supplied by placing a beaker of water in the incubator during the growth period. A moist environement retards dehydration of the medium and thereby avoids misleading experiemental results.

Shaker waterbath

Thermostatically controlled shaking waterbath is another piece of apparatus used to cultivate microoganisms. Its advantage is that it provides a rapid and uniform transfer of heat to the culture vessel, and its agitation provides increased aeration, resulting in acceleration of growth (only in broth medium).

Refrigerator

prevent dehydration-- also, a repository for thermolabile solutions, antibiotics, serums, and biochemical reagents.

Culture Transfer Techniques

Learning objections: 1. technique for aseptic removal and transfer of microbes for subculturing. 2. Correctly sterilize inoculation instruments in the flame of a Bunsen burner. 3. Correctly manipulate your fingers to remoce and replace the test tube closure.

Principle: microorganisms are transferred from one medium to another by subculturing.

microbes must be transferred from one vessel to another or from stock cultures to various media for maintenance and study called subculturing and must be carried out aseptically. Microbes are alwasys present in the air and on lab surfaces, benches, and eqquipment--a source of contamination and interfere with experimental results unless proper aseptic techniques are used during subculturing figure (1). 1. Label the tube with name of organism and your initials. 2. hold the stock culture tube and the tube to be incoulated in the palm of your hand, secure with our thumb, and separae the two tubes to form a V in your hand. 3. Sterlize an inoculation needle or loop by holding it in the hottest portion of the Bunsen burner flame, until the wire becomes red hot.. Then, rapidly pass the upp portion of thehandle through the flame. Once flamed, the loop is never put down but is held in the hand and allowed to cool for 10 to 20 seconds. 4. Uncap the tubes by grasping the first cap with your little finger and the second cap with your next finger and lifting the clsure upward (never place down on bench). 5. After removing the closure, flame the necks of the tubes by briefly passing them through the flame. The sterile tranfer instrucment is further cooled by touching it to the stierile inside wall of the culture tube before removeing a small sample of the inoculum. 6. Use a loop or needle for removal of inoculum. do not gouge the agar.a needle is always used when transferring microorganisms to an agar deep tube broh solid andliquid cultures.A. for slant-to- broth, lightly shake the loop or needle in the broth culture to dislodge the microorganisms. B. for broth-to-slant transfer, obtain a loopful of broth and place at the base of an agar slant medium. lightly draw the loop over the hardened surface in a straght or zigzag line, from the base of the agar slant to the top. C. slant-to-agar-deep transfer, obtain the inoculum from the agar slant. insert a straight needle to the bottom of the tube in a straight line and rapidly withdraw along the line of insertion. this is called a stab inoculation. 7. Following incoulaiton, remove the instrument and reflae the necks of the tubes. 8. replace the caps on the same tubes from which they were removed. 9. reflame the loop or needle to destroy any remaining organsms. in this experiment you will master aseptic transfer.

AT THE BENCH: materials

CULTURALS-- 24 hour nutrient broth and nutrient agar slant cultures of Serratia marcescens. MEDIA-- one nutrient broth, one nutruient agar slant, and one nutrient agar deep tube. EQUIPMENT--Bunsen burner, incoculation loop and needle, and glassware marking pencil.

PROCEDURE LAB ONE:

1. label all tubes of sterile media as described in the lab protocol. 2. following he procedure outlined andillustrated previouly (figure 1) perform the following transfers: A. S. marcescens broth culture to a nutrient agar salnat, nutrient agar deep tube, and nutrient broth. B. S. marcescens agar slant culture to a nutrient broth, nutrient agar slant, and nutrient agar deep tube. 3. incubate all cultures at 25°C for 24 to 48 hours.

PROCEDURE LAB TWO

1. examine all culture for the appearance of growth, which is indicated by turbidity in the broth culture and theappearance of an orange-red growth on the surface of the slant and along the line of inoculaito in the agar deep tube. 2. record yur observations in the chart provided in the lab report.

REVIEW QUESTIONS: Explain why the following steps are essential during subculturing a. flaming the inoculation instrument prior to andafter each inoculation

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Explain why the following steps are essential during subculturing b. holding the test tube caps in the hand

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Explain why the following steps are essential during subculturing. c. cooling the inooculation instrument pror to obtaining the inoculum.

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Explain why the following steps are essential during subculturing .d. flaming te neck of the tubes immediately after uncapping and before recapping.

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2. Describe the purposes of the subculturing procedure.

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3. Explain why a straight inoculation needle is used to inoculate an agar deep

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4.there is a lackof orange-red pigmentatio in some of the growth on your agar slant labeled S. marcescens. Does this necessarily indicate the presence of a contaminat? explain.

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Upon observation of the nutrient agar slant culture, you strongly suspect that the culture is contaminated. Outline the metod you would follow to ascertain whether your suspicion is justified.

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