lab final bacterial transfers & Oxygen requirements & growth patterns

what are the proper steps for a sterile transfer?

1. Label the sterile media tube with yur name, date, and organism name

2. Turn on Bacti- Cinerator and allow it to heat for at least 10 minutes

3. sterilze the inoculation loop by inseting it into the cinerator until it glows red (about 10 sec)

4. cool the loop for a few seconds to avoid killing the bacteria or causing aerosols 

5. Hold both tubes (stock and sterile) in one hand- stock culture closest to you 

6. Loosen both caps but do not set them down; hold them with your fingers

7. flame the tops of both tubes by briefly passing them through the cinerator opening 

8. obtain the inoculum by dipping the cooled loop into the stock culture 

9. transfer the inoculum into the sterile broth by gently touching or swirling the loop in the liquid 

10. flame the tube openings again, then replace the caps loosely 

11. re- sterilize the loop before settiing it down 

12. incubate the inoculated tubes 37 degrees celsius for 48 hours 

what are the importance of aseptic technique?

• prevents contamination of the culture with unwanted organisms

• prevents spread of microorganisms to lab personnel and the environment  

• Ensures that results are accurate and reliable, reflecting growth of the intended organism only

what is a pure culture?

a culture containing only one species of microorganism 

what is sterile culture (sterile medium)?

contains no living organisms at all - completely free of microbes before inoculation

what is mixed culture?

contains two or more intentionally grown species of microorganisms

what is contaminated culture? 

a culture that has been invaded by unwanted microorganisms or foreign material

name some specific things one can do to prevent contamination

1. Flame or heat - sterilize all tools (loops, needles, tube mouths) before and after each use

2. Keep tube caps in your hand - never place them on the lab bench

3. Avoid talking, coughing, or sneezing over open cultures

4. Label everything clearly before beginning any transfer

5. Work near a heat source (like a Bacti-Cinerator) to create an updraft that minimizes airborne contamination

6. Cool the loop before touching the culture to avoid creating aerosols or killing bacteria

7. Do not touch sterile surfaces (tube rims, loops tips, agar surfaces) with fingers or other non-sterile items

8. Clean the workspace with disinfectant before and after the lab session

9. Practice careful handling - no rushing or unnecessary movements while tubes are open

what are the two main goals of aseptic (sterile) technique?

1. prevent contamination of the culture

2. prevent spread of microorganisms to people or the environment

why is it important to always treat all microorganisms as potential pathogens?

because in large enough numbers or through the right portal of entry, almost any bacterium can cause infection

what is the purpose of heat-sterilizing (flaming) the inoculation loop?

to kill all microorganisms on the loop and prevent contamination

why should you never set test tube caps on the lab table?

to avoid contaminating the caps and the work surface

what happens if the inoculating loop is too hot when touching the culture?

It can kill the bacteria and create aerosols that contaminate the environment 

what does the term inoculum mean?

the small sample of organisms transferred from one culture to another

what is a growth medium?

a nutrient- rich substance that provides food and conditions for microorganisms to grow

what are obligate aerobes?

bacteria that require oxygen (O2) to grow. Growth appears only at the top of the broth tube 

what are facultative anaerobes?

bacteria that can grow with or without oxygen, but grow best near the top where 02 is present

What are aerotolerant anaerobes?

Bacteria that don’t use oxygen but aren’t harmed by it. Growth is evenly distributed throughout the tube

what are obligate anaerobes?

Bacteria that cannot survive in the presence of oxygen; growth occurs only at the bottom of the tube 

What is the main purpose of the streak plate technique?

to isolate individual bacterial colonies from a mixed culture by gradually spreading out bacteria across the agar surface

what are the three organisms commonly used for this procedure in lab?

E.coli, S.epidermidis (S.epi), and Bacillus subtilis (B.sub)

What are information must be written on the agar plate before beginning the procedure? 

Student’s name, date, and the organism name - written on the bottom of the plate 

Why should the Petri dish lid stay mostly closed during streaking?

To prevent airborne contaminants from falling onto the agar surface

What tool is used to transfer bacteria in the streak plate method?

an inoculating loop, which must be heat- sterilized between each streak section

Why is heat-sterilizing the loop between streaks important? 

It dilutes the bacterial sample, ensuring fewer bacteria are transferred each time to help isolate single colonies 

How is section 1 streaked on the agar plate?

The loop is gently swept back and forth across section 1 without digging into the agar

When streaking section 2, where do you draw bacteria from?

from section 1 into section 2 - without overlapping previous streaks within section 2

When streaking section 3, what section should NOT be re-entered? 

Section 1 - only streak from section 2 into section 3 

What is the goal of streaking across three sections?

to dilute bacteria enough that isolated colonies form in section 3 after incubation

what is a bacterial colony?

A visible cluster of bacteria grown from a single cell - a clone of identical cells

At what temperature and time should plates be incubated? 

37 degree celsius for a minimum of 48 hours 

Why are agar plates stored inverted during incubation?

To prevent condensation from dripping onto the agar, which could spread bacteria and distort results

why is agar used as a growth medium? 

agar provides nutrients and a stable surface for bacteria to grow while remaining solid at incubation temperatures 

What is the general purpose of inoculating an agar plate (not streaking)?

To allow general bacterial growth without isolating colonies - often done with a sterile loop or swab

Why should the agar not be dug into during streaking?

Damaging the agar can trap bacteria or cause uneven growth, interfering with colony isolation

what happens if the loop isn’t cooled before touching the agar? 

The heat can kill the bacteria or damage the agar surface 

What does a successful streak plate look like?

clear, isolated colonies in section 3 with progressively fewer colonies in sections 1 and 2