Module 03: Aseptic Techniques

Question 1

In working with microorganisms, what is entailed for the biologist to do?

Answer 1

In working with microorganisms, we must have a method of transferring growing organisms (called the inoculum) from a pure culture to a sterile medium without introducing any unwanted outside contaminants.

Question 2

This is known as the growing organisms transferred from a pure culture to a sterile medium without introducing any unwanted outside contaminants.

Answer 2

Inoculum

Question 3

Anything in or on which we grow a microorganism is termed a ________________.

Answer 3

Medium or culture medium.

Question 4

This is one which is free of all life forms. It is usually sterilized by heating it to a temperature at which all contaminating microorganisms are destroyed.

Answer 4

Sterile Medium

Question 5

This is delineated as the method of preventing unwanted microorganisms from gaining access.

Answer 5

Aseptic Techniques

Question 6

What are the different types of media (based on consistency)?

Answer 6

(1) Broth
(2) Semisolid
(3) Solid

Question 7

This type of media based on consistency lacks a solidifying agent such as agar.

Answer 7

Broth

Question 8

This type of media based on consistency has an agar concentration less than 1%?

Answer 8

Semisolid

Question 9

This type of media based on consistency has an agar concentration of 1.5% to 1.8%.

Answer 9

Solid

Question 10

What are the types of media based on consistency under solid?

Answer 10

(1) Agar Slant
(2) Agar Deep (Butt)
(3) Agar Plate

Question 11

This is known as a polysaccharide derived from seaweed.

Answer 11

Agar

Question 12

When does agar liquify or solidify?

Answer 12

It liquifies at 100°C and solidifies at 40°C.

Question 13

How is an Agar Slant prepared?

Answer 13

7 ml per 16 x 125 mm tube gives a slanted surface with good surface area and a “butt” about 1 inch deep

Question 14

This is an isolation procedure used to isolate bacteria from a mixed bacterial culture.

Answer 14

Streak Plate Method

Question 15

How is a streak plate method executed?

Answer 15

It is a dilution technique in which a bacterial sample is streaked over the agar surface in quadrants so that bacterial cell density gets diluted and individual cells spread over the agar surface.

Question 16

What is the result from executing a streak plate method?

Answer 16

(1) These individual bacterial cells grow and form isolated colonies.
(2) An isolated colony is further transferred in a separate sterile medium and pure culture is obtained.

Question 17

What are the advantages of the streak plate method?

Answer 17

(1) Streak plate techniques are the most popular and widely used procedure of isolating specific bacteria from a mixed population of bacteria.
(2) Streak can be performed in many patterns.
(3) It is the first step in the identification study of any microorganism.
(4) Streak plate method is a less time-consuming method.
(5) Heat sensitive organisms are not affected.
(6) Aerobic microbes grow easily.

Question 18

What are the limitations of the streak plate method?

Answer 18

(1) There are high chances of contamination while streaking.
(2) This method is not suitable for the growth of anaerobes.
(3) This is qualitative method and not suitable for microbial enumeration.

Question 19

This type of microbial culture is composed of two or more species of microorganism or bacteria present.

Answer 19

Mixed Culture

Question 20

This type of microbial culture is composed of single species of microorganism or bacteria present.

Answer 20

Pure Culture

Question 21

How is broth transfer executed?

Answer 21

(1) Label the tube to be used with the microorganism used, the date, and your name or initials.
(2) Take in one hand the Serratia broth culture and one labeled tube of sterile broth.
(3) Take the inoculating loop with the other hand and flame the entire wire portion to redness.
(4) Remove the plugs or caps from the tubes by grasping them between the fingers of the hand holding the inoculating loop.
(5) Flame the mouth (lips) of both tubes
(6) Insert the loop into the broth culture and obtain a loopful of culture.
(7) Introduce the inoculum by immersing the loop into the sterile broth. (Before removing the loop, touch it to the inner tube surface to eliminate excess broth).
(8) Flame the lips of both tubes again and replace the plugs or caps in their respective tubes.
(9) Flame the inoculating loop again and put it down on a metal surface. Incubate tubes as directed.

Question 22

How is the procedure of agar slants as sources of inoculum executed?

Answer 22

(1) Label the tube with the microorganism to be used, the date, and your name or initials.
(2) Take in one hand the agar slant Micrococcus culture and one tube of sterile broth. Hold the agar slant so that it faces you.
(3) Take the inoculating needle with your other hand and flame the wire portion to remove redness.
(4) Remove the plugs or caps from the tubes by grasping them between the fingers holding the inoculating needle.
(5) Flame the lips of both tubes.
(6) Obtain an inoculum by removing a small portion.
(7) Immerse the inoculum in the broth and gently shake the needle to free the microorganisms sticking to it.
(8) Flame the lips of both tubes again and replace the plus or caps in their respective tubes.
(9) Flame the inoculating needle and put it down in a suitable place. Incubate as directed.

Question 23

How is the procedure of the inoculation of an agar slant executed?

Answer 23

(1) Label the agar slant with the organism to be used, the date and your name or initials.
(2) Take in one hand the Serratia broth culture and one sterile nutrient agar slant. Hold the slant surface so that it faces upward and toward you.
(3) Take the inoculating loop with your other hand and flame the entire wire portion to redness.
(4) Remove the plugs or caps from the tubes by grasping them between the fingers of the hand holding the inoculating loop.
(5) Flame the lips of both tubes.
(6) Obtain the inoculum by removing a loopful of the broth culture.
(7) Place the loop into the sterile agar slant’s surface at its bottom. Move the loop from side to side as you pull it upward out of the tube. Do not dig into the agar.
(8) Flame the lips of the tubes and replace the plus or caps.
(9) Flame the inoculating loop and place it on an appropriate surface. Incubate as directed.

Question 24

How is the procedure of colony selection performed?

Answer 24

(1) Label the tube with the microorganism to be used, the date, and your name or initials.
(2) Select an isolated Bacillus colony and circle it with a wax pencil on the petri plate’s bottom.
(3) Flame the inoculating needle to redness.
(4) Lift the Petri plate top, cool the needle by touching a clear agar surface and remove a small amount of growth from the circle colony. Close the colony.
(5) Remove the plug or cap from one tube of sterile broth and flame the lip of the tube.
(6) Immerse the inoculum in the broth and shake the needle to free the microbial growth sticking to it.
(7)Flame the lip of the tube and replace the plug or cap.
(8) Flame the inoculating needle and put it down on an appropriate surface. Incubate as directed.

Question 25

How is the pour plate technique executed?

Answer 25

(1) Place nutrient agar deeps into the boiling water bath for melting.
(2) Remove a melted deep, cool to 45°C (this temperature can be tolerated on the inner surface of the forearm), and wipe the tube to remove any moisture.
(3) Take the tubes of cool melted. agar and the mixed culture in one hand. Flame the inoculating loop and the lips of the tubes after removing their plugs or cap.
(4) Remove one loopful of the culture and inoculate the melted deep.
(5) Flame the lips of both tubes, replace the plugs or caps correctly, and put the tubes down. Flame the loop and put it down.
(6) Pick up and gently shake the freshly inoculated deep. Remove the plug or cap and flame the lip of the tube.
(7) Pour the contents of the tube into the bottom of a labeled Petri dish by slightly raising the dish’s top.
(8) Flame the lip of the tube, replace the plug, and dispose of the contaminated tube as directed
(9) Rotate the plate gently to dis- tribute the agar evenly, and allow the agar to harden (about 10 min).

Question 26

How is the streak plate technique performed?

Answer 26

(1) Shake the mixed culture tube gently.
(2) Flame the inoculating loop to redness, remove the plug or cap of the tube with the free fingers of the hand holding the inoculating loop. and flame the lip of the tube.
(3) Remove a loopful of the mixed culture after the loop has cooled for at least 5 seconds.
(4) Flame the tube once again, replace the plug or cap, and put the tube in a rack or another appropriate place.
(5) Hold the Petri plate so that the bottom rests on the palm of the left hand if you are right-handed or the right if you are left-hand. Lift the Petri plate cover and place the inoculum at the edge of the agar surface farthest from you.
(6) Streak the inoculum on the agar surface from side-to-side in parallel lines covering approximately one quarter of the plate and in the pattern shown. Do not dig into the agar.
(7) Lower the top off the plate and flame the inoculating loop. Rotate the Petri plate one quarter of a full turn.
(8) Lift the dish top and cool the inoculating loop by touching the agar surface away from the set of streaks. Skim the inoculating loop over the first set of streaks, once, and make a second set of streaks. Lower the plate top
(9) Repeat steps 7 and 8. The final result should be a set of distinct streaked regions as shown. Flame the inoculating loop before put ting it down. Incubate the plate in an inverted position.

Question 27

What is the four way streak (quadrant streak) and their areas of growth)?

Answer 27

(1) Quadrant A (Area 1 - Heavy Confluent growth)
(2) Quadrant B (Area 2 - Less Dense growth)
(3) Quadrant C (Area 3 - Weak Growth)
(4) Quadrant D (Area 4 - Isolated Single Colonies)

Question 28

This is a plate which has been streaked showing the colonies thinning as the streaking moves clockwise.

Answer 28

Four-way Streak (Quadrant Streak)

Question 29

This is known as the three sector method.

Answer 29

T-streak

Question 30

What is the three sector streak (T streak) and their areas of growth)?

Answer 30

(1) First Sector - Confluent Heavy growth
(2) Second Sector - Less Heavy Growth but no isolated colonies
(3) Third Sector - Isolate colonies

Question 31

What are the different types of streaking methods?

Answer 31

(1) Zigzag streak
(2) Radiant Streak
(3) Continuous Streak

Question 32

What should be labeled in your agar plate prior to streaking?

Answer 32

(1) Type of Microorganism
(2) Date
(3) Agar Type
(4) Initials