Potential Risks and Safety - Serial Dilations

Potential Risks and Safety Procedures:

  • Always be aware of potential hazards in the lab.

  • Familiarity with risks and safety protocols reduces accident potential.

  • Labs with hazardous materials must display the universal biohazard symbol.


Chemical Hazard Labels:

  • can be diamond-shaped or large rectangles.


Color-coded:

Blue - health risks

Red - flammability risks

Yellow - reactivity risks

White - Specific risks


Numbered 0-4:


0 = Minimal risk

1 = Slight risk

2 = Moderate risk

3 = Serious risk

4 = Extreme risk


Safety Data Sheet (SDS)

  • contains more specific information about the chemical


Biosafety Levels:

BSL-1:

  • Microorganisms are non-pathogenic to healthy adults.

  • Standard aseptic techniques are sufficient.


BSL-2:

  • Microorganisms may be slightly pathogenic or environmentally harmful.

  • Requires standard aseptic techniques and autoclaving contaminated materials.

  • Biohazard signs on doors, restricted access.

  • Use of personal protective equipment (PPE): goggles, gloves, lab coat.



Standard operating procedures (SOP):

  • Food and drink are not allowed 

  • Proper clothing is required for safety reasons and includes no open-toed shoes, no shorts, no sleeveless shirts, and long hair must be tied back. 

  • Goggles need to be worn unless you wear glasses. 

  • Gloves will be provided. 

  • A lab coat is not required, but we suggest wearing a large shirt over your everyday clothes to help keep them clean and not get contaminated.  

  • The lab safety contract will be reviewed by your instructor on the first day, so you are familiar with all safety rules and protocols.


Microorganism Cultures and Aseptic Techniques

Purpose of the lab testing is to determine what the causative agent.


Types of Media:

  1. Broth to broth: using the aseptic technique(heat inoculating loop till glowing > cool it a bit > carefully open the tube > pass mouth to the flame > immerse loop in the specimen> remove loop, pass mouth of the tube, then close > open the other sterile tube > flame mouth > insert inoculating loop > swirl to inoculate > remove loop > pass mouth of tube > heat inoculating loop)

  • transfer a loopful of the bacteria from the stock tube to the sterile tube. 

  • Make sure your tube is correctly labeled before placing it in the appropriate incubator. 

  1. Broth to agar: using the aseptic technique(heat inoculating loop till glowing > cool it a bit > carefully open the tube > pass mouth to the flame > immerse loop in the specimen> remove loop, pass mouth of the tube, then close > open the other sterile tube > flame mouth > insert inoculating loop >  touch the end of the loop or needle on the surface of the agar and smear it to spread it on the surface to inoculate it > remove loop > pass mouth of tube > heat inoculating loop)

  •  transfer a loopful of the bacteria from the stock tube to the nutrient agar slant. Using the loop, move it in a zigzag fashion across the surface of the slanted agar.

  •  Label the tube and place it in the appropriate incubator. 

  1. Agar to broth: using the aseptic technique(heat inoculating loop till glowing > cool it a bit > carefully open the tube > pass mouth to the flame > scrap a little on the end of the instrument > remove loop, pass mouth of the tube, then close > open the other sterile tube > flame mouth > insert inoculating loop > swirl to inoculate > remove loop > pass mouth of tube > heat inoculating loop)

  • use a loop to scrap a little of the bacterial specimen from the surface of the stock agar tube and transfer it to the broth.

  • Label the broth tube and place it in the appropriate incubator. 

  1. Agar to agar: using the aseptic technique(heat inoculating loop till glowing > cool it a bit > carefully open the tube > pass mouth to the flame >  scrap a little on the end of the instrument > remove loop, pass mouth of the tube, then close > open the other sterile tube > flame mouth > insert inoculating loop >  touch the end of the loop or needle on the surface of the agar and smear it to spread it on the surface to inoculate it > remove loop > pass mouth of tube > heat inoculating loop)

  • use a loop to scrap a little of the bacterial specimen from the surface of the stock agar and transfer it to the new agar slant by moving the loop in a zigzag across the surface of the agar slant surface. 

  • Label the new tube and place it in the appropriate incubator. 


⭐ All of the tubes from steps 1-3 will be incubated at 37°C for 24 hours. 

⭐ The tube from step 4 will be incubated at room temperature for 24 hours.

⭐ After the incubation period, observe all of the tubes for growth. If bacteria are growing in a liquid medium, it will turn turbid (cloudy).

⭐ Notice if there is a difference in pigmentation depending on the incubation temperature.


Liquid Culture:

  • Growth appears as turbidity (cloudiness).

  • Fungi look like small spheres.

Solid Media:

  • Made by adding agar to liquid media.

  • Can be used in petri dishes, slants (in test tubes to increase surface area), or deep tubes (stabbed for growth).


Choice between solid and liquid media depends on the experiment:

⭐ liquid is usually for maintaining pure cultures

⭐ solid is for stock cultures, physiological tests, isolation, and studying diversity.


Types of Culture Transfer:

  • Broth to Broth Liquid to liquid

  • Broth to AgarLiquid to solid

  • Solid to liquid

  • Solid to solid


Tools Used:

  • Inoculating loop

  • Inoculating needle

  • Pipette


Techniques:

  • Use aseptic techniques to prevent contamination.

  • Transfer fungi using specific techniques, discussed later.


Aseptic Techniques:

Importance: Prevents contamination of samples and protects personnel.

Safety Zone: Always work within 6-8 inches of the flame for sterility.

Sterilizing Instruments:

  • Flame the inoculating loop/needle until glowing and let cool.

  • Avoid immediate use of media to prevent killing the specimen.

Handling Test Tubes:

  • Open near the flame, pass the mouth through the flame, and then insert the loop/needle.

  • Re-flame the mouth of the tube before capping.

  • Handling Petri Dishes:

  • Flame loop/needle, let cool, then open the dish to scrape the specimen.

  • Keep the dish close to the burner to maintain sterility.

Using these techniques ensures that cultures remain uncontaminated, providing reliable results for various microbiological experiments. Perfect for setting a solid foundation in laboratory practices.


Pour Plate Method


Obtain a pure culture and calculate the number of microorganisms in a sample, such as soil.


⭐ Microorganisms can grow within the media and on the surface, benefiting those with lower oxygen requirements.


Procedure:

  • Melt Agar: Heat the media in a hot water bath (melt at 100°C, solidify at 45°C).

  • Cool Media: Remove and cool the media slightly to avoid killing the specimen.

  • Inoculate Media: Aseptically remove a sample from a 24-hour broth culture using a sterile inoculating loop. Inoculate the first tube (A), mix, sterilize the loop, and use tube A to inoculate tube B. Mix tube B, sterilize the loop, and use tube B to inoculate tube C.

  • Pour Media: Pour contents of each tube into separate sterile petri dishes, swirl to cover the bottom evenly.

  • Solidify and Incubate: Allow media to solidify, then invert the plates and incubate at 37°C for 24 hours.

  • Observation: After incubation, observe the growth on each plate.


Notes:

  • Serial Dilution: Often combined with serial dilution for different concentrations.

  • Storage: Store plates inverted to prevent condensation on the agar surface.


Using the pour plate method provides a robust way to isolate and count microorganisms, especially those that thrive in lower oxygen environments. It's critical to maintain proper temperature and aseptic conditions to ensure accurate results. This technique is useful for both quantitative and qualitative analysis in microbiological studies



Using Pipettes and Micropipettes

Types of Pipettes:

⭐ Micropipettes:

  • Used for very small volumes (1.0 ml or less).

  • Calibrated for specific volumes within a certain range.

  • Preset volume or adjustable via a dial.

  • Use aseptic technique to attach the tip, draw up the liquid, and dispense it accurately.

  • Dispose of tips in the regular trash for nonhazardous liquids or biohazard containers for microorganisms.


⭐Serological Pipettes:

  • Used for volumes of 0.1 ml and larger.

  • Available in 1.0, 5.0, 10.0, 25.0, and 50.0 ml sizes.

  • Individually wrapped in sterile wrappers.

  • Attach to a mechanical pipette pump to draw and dispense liquid.

  • Measure from the bottom of the meniscus for accuracy.


🚨Dispose of in the regular trash if used for water, or biohazard containers if used for live microorganisms.


Aseptic Technique:

  • Prevents contamination of samples and ensures the safety of personnel.

  • Always work within 6-8 inches of a flame for a sterile environment.

  • Sterilize the pipette or pipette tip before use and handle all samples carefully to avoid aerosols.

  • Proper disposal of pipettes and tips based on the type of liquid used.


Steps for Using Serological Pipettes:

  • Select the appropriate size for the required volume.

  • Attach a mechanical pipette pump to the pipette.

  • Draw up the liquid by rotating the dial on the pump.

  • Measure from the bottom of the meniscus.

  • Dispose of the pipette appropriately based on the liquid type.


Steps for Using Micropipettes:

  • Set the specific volume using the dial.

  • Attach the tip using aseptic technique.

  • Draw up the liquid by depressing the plunger to the first stop, then raising it.

  • Dispense the liquid by moving to the receiving tube or flask, depressing to the second stop.

  • Remove the tip from the liquid before releasing the plunger to avoid re-drawing liquid.

  • Dispose of the tip appropriately based on the liquid type.


By following these steps and maintaining aseptic techniques, you ensure accurate measurements and prevent contamination in your experiments. It's essential to handle each step with care to achieve reliable results in any scientific research.


Serial Dilutions


Determine microorganism concentration for food safety and water quality.

Helps in taking precautions if bacterial concentration is too high.


Method:

  • When to Use: Original sample has a high concentration of bacteria.

  • Plate Counting Range: Count colonies within 30 to 300 range; ignore counts less than 30 (TFTC) or more than 300 (TNTC).


Procedure:

Prepare Serial Dilutions:

  • Use sterile tubes with 9.0 ml of water.

  • Perform consistent dilutions, usually 10-fold.


Dilution Steps:

  • Tube A: Add 1.0 ml of the original sample to 9.0 ml water (1:10 dilution).

  • Tube B: Transfer 1.0 ml from Tube A to another 9.0 ml water tube (1:10 dilution of A, overall 1:100).

  • Continue the process as needed.


Plating and Counting:

  • Plate samples on nutrient agar.

  • Count colonies within the 30-300 range.

  • Multiply the number of colonies by the dilution factor to determine the original concentration.


Key Notes:

Confluent Growth: Solid growth without individual colonies (lawn).

Accuracy: Ensure proper dilution to achieve countable colonies within the desired range.

Overall Dilution Calculation: Multiply all dilution factors used (e.g., Tube A 1:10 and Tube B 1:10 = 1:100).


By following these steps, you can accurately determine the concentration of microorganisms in your sample, crucial for food safety and water quality testing. Proper serial dilution ensures reliable and precise results.