Aseptic Technique and Streaking Plates

Aseptic Transfers

Aseptic transfers involve moving living microbes from one location to another without contaminating the culture, sterile medium, or surroundings.
Essential considerations:
- Source of the sample.
- Type of transfer instrument required.
- Destination of the sample.
Sterilization: Inoculating instruments are sterilized to prevent contamination.
- Sterilization is the process of removing all microorganisms and pathogens from an object or surface.
- Methods include chemical treatments, high heat, or radiation.

Sterilization Methods

Inoculating loops and needles:
- Sterilized using a Bunsen burner or microincinerator immediately before use.
- The mouths of tubes or flasks are also passed through heat to eliminate contaminants.
Instruments not suitable for flaming:
- Plastic Pasteur pipettes, cotton applicators, glass pipettes, and micropipettor tips.
- Sterilized inside wrappers or containers by autoclaving well in advance of use.
Autoclaves:
- Utilize high pressure with steam at $121^\circ C$ ( $249^\circ F$ ) for 15–20 minutes.

Basics of Aseptic Transfer

Organization: Arrange all media/slides beforehand and label everything clearly.
Patience: Work efficiently but carefully, given potential hazards.
Microincinerator: Preheat on high setting for 15-20 minutes.
Inoculating Loop Handling: Hold like a pencil in the dominant hand, wire part facing away.
Loop Sterilization:
- Incinerate the metal loop in the microincinerator.
- Insert into the hub (opening) up to the handle for about 10 seconds without touching the sides.
- The loop will likely turn orange/red.
Cooling:
- Remove without touching the incinerator walls and hold near the opening to cool for ~10 seconds.
Sterile Loop Caution: Avoid touching the sterile loop on any surface or waving it in the air.
Culture Tube Handling:
- Hold the culture tube in the non-dominant hand.
- Open the lid with the same hand, holding it at an angle to prevent airborne contamination.
- Insert the sterile loop to collect the sample, positioning it just below the liquid's surface to capture a droplet.
- Avoid touching the tube's sides when withdrawing the loop.
Immediate Closure: Promptly close the tube lid after removing the sample.
Immediate Transfer: Transfer the bacteria to its destination promptly.
- (a) For broth: Remove the tube lid and insert the inoculating loop without touching the sides, positioning it just below the liquid's surface. Gently swish the loop before removing it.
- (b) For agar: Gently touch the loop onto the agar surface without gouging it, and gently move the loop over the surface. Promptly close the plate lid afterward.
Loop Sterilization (Repeat): Sterilize the inoculating loop between each sample transfer and after completion. Avoid placing a dirty loop on the bench; sterilize after every use.
Transferring Colonies to Slides
- Sterile droplet of water first needs to be placed on the slide
- Aseptically transfer the colony to the droplet of water
- Make sure that the colony is dispersed in the water droplet as much as possible.
- Larger, thicker chunks of the colony might retain the dyes used in staining.

Media

Growth medium or culture medium is a solid or liquid designed to support the growth of microorganisms or cells.
Nutrient Agar:
- Contains simple ingredients suitable for the growth of many organisms.
- Supplied as a powder, which is measured, added to water, and boiled for ~1 minute to ensure a uniform solution.
- After autoclaving, the molten nutrient agar is then cooled to about $50 ^\circ C$ and poured into sterile plastic Petri dishes.

Sterilization of Media

Like all bacterial media, the nutrient agar is then autoclaved to sterilize it.
Heated to $121^\circ C$ for at least 15 minutes using steam and pressure to eliminate bacteria and endospores.
This ensures that only intentionally introduced bacteria grow in the media.

Post-Autoclaving Procedures

Molten nutrient agar is cooled to approximately $50^\circ C$ and poured into sterile Petri dishes, which are immediately covered to maintain sterility.
Once solidified (about 30 minutes), plates are stored upside down, often refrigerated until use.
Upside-Down Storage: Prevents condensation from dripping onto the agar, which can disrupt colony formation and spread bacteria.
When needed, plates are removed from refrigeration ( $4^\circ C$ ) and allowed to warm to room temperature (about $25^\circ C$ ).

Inoculation

The act of introducing microorganisms into/onto culture media

Streaking for Isolation

A technique to isolate individual bacterial colonies for obtaining a pure culture.
A single bacterial colony is a visible mass of microorganisms originating from a single mother cell.
Colonies are clusters of millions of genetically identical bacteria (clones) from one cell.

Streaking Procedure

Materials Needed:
- Inoculating loop
- Microincinerator
- Marker
- Bacterial culture (broth or agar)
- Sterile nutrient agar plate
Plate Labeling:
- On the bottom of the plate, write your initials, sample name, and date along the outside edge.
Streaking Guidelines:
- Open the plate only when inoculating.
- Avoid leaving the plate open to prevent contamination.

Streaking Steps

Zone 1:
- Aseptically remove a small amount of bacteria and gently streak it across one quarter of the plate using side-to-side motions.
- Flame the loop.
Zone 2:
- With a sterilized loop, drag the loop 2-3 times into zone 1.
- Create a new streak section on another quarter of the plate.
- Flame the loop.
Zone 3:
- With a sterilized loop, drag the loop 2-3 times into zone 2.
- Create a new streak section on another quarter of the plate.
- Flame the loop.
Final Zone:
- With a sterilized loop, drag the loop 2-3 times into zone 3.
- Create a new streak section on the final quarter of the plate.
- Flame the loop.
Incubation:
- Place the labeled plate upside down in a $25^\circ C$ incubator.
- Upside-down placement prevents condensation from smearing bacteria.
Growth Period:
- Allow bacteria to grow for 2 days (48 hours) before examination.