Microbiology Lab Essentials :

Microbiology Lab Essentials :

✓ Slant Preparation & Streaking : 

a)Agar  Slant  Tube Preparation

1)  Calculate  the  amount  of  media  that  needs  to  be  made. o  Each broth  tube  requires  7-10mL of  broth. o  If 100 tubes  are  needed, 700-1000mL of  broth  is  needed. o  Always add  200mL to the amount  required in case of  spills  or miscalculation.

2) Follow package instructions  for preparation.    o  Instructions  are typically  written for 1L (1000mL) of  media.   If  less  is  desired  calculate  the amount  needed  as  shown:

3)  For example:   If  the  instructions  state 23g  for 1L  and  600mL is  desired,  use a  ratio to  calculate the amount  needed  (in this  example 13.8  g is  needed  for preparing 600mL): 

4) 23g/1000mL =  Xg/600mL  23 * 600=  1000X  

13800  = 1000X   13800/1000  =X 

13.8  g  =  X o  Always prepare  media in a  beaker with  1/3  of  empty  space.

(i.e.  prepare  600mL of  media in a 1000mL beaker).  

5) If  the amount  of  media to  be  prepared  is  greater  than  1L,  prepare it  in 500mL aliquots  or use  a 2000mL beaker.

6) Label the  beaker with  autoclave tape  and  state  what  media is  being  prepared,  the  date,  and your  initials  (i.e.  Nutrient  Broth    8-18-08 KH).

7) Add powder to  beaker first,  and then  fill  with necessary  amount  of  water. o  Stir  with  a  glass  stirring rod  to  mix. o  Place  in  microwave and  heat  at  3-5 minute  intervals. 

8) Stir  between  intervals, using caution  and  allowing the  media to sit for 30  seconds  in the microwave  before stirring.

9) Heat for approximately  10  minutes  or until  boiling  has  been  achieved. 

10)  While heating,  place test tubes  in  racks  and label  with  autoclave tape  (i.e.  Nutrient  Agar Slant's  8-18-08  KH). 

11) Test the pH of  the  media to  insure that  it  is  within the acceptable range  as  stated  on  the package. If  the pH needs  to be  adjusted, add drops  of  1N Hydrochloric  Acid (HCl)  (to  make more acidic)  or  1N  Sodium  Hydroxide  (NaOH)  (to  make more basic)  as  necessary  until desired  pH  is  achieved.

12) Pour the  broth  into an appropriately  sized glass  bottle  for pipette dispenser  use.

13)  Before attaching  the  pipette  dispenser, set  it to  the  proper  setting  for the volume of  media required for each tube.

14) Attach  the  dispenser to  the  bottle top. While over the sink, test the dispenser to ensure  that  the  liquid  media is  filling  the  pipette dispenser.

15) Fill  each  tube Place  caps  on filled  tubes. For screw  caps,  leave the caps  partially  unscrewed  to  allow  steam  to  enter and escape.Autoclave for 20 minutes. 

16)  Refer to the  Standard Operating Procedure  for Autoclave Use.After sterilization  is  complete, remove the test tube  rack, tighten  all  test  tube caps,  and  tilt the  tubes  of  liquid  agar  on  a support that is  about  ½  inch thick  (Plastic  weighing  dishes  work well)  or use special  white  “slant  racks”. 

17) Allow the  agar to solidify  (about  25  minutes)  and  store  in the  refrigerator.

Pick up a loopful of your inoculum from either a broth or an agar culture. Using a sterile agar medium plate (lift the lid just enough to insert the loop), streak a vertical line straight down.

When streaking the agar, keep the loop horizontal and only streak the surface of the agar: DO NOT DIG INTO THE AGAR.

Move the loop in a zig-zag pattern across the agar until 1/3 of the Slant is covered, finishing the first section.

Sterilize the loop in the flame and let it cool before continuing to spread the bacteria. You can do this by 

1) sticking the hot loop in the agar at the edge of the agar away from the bacteria, or

2) just holding the loop for a few seconds while it cools.the bacteria from the first streak into using the same zig-zag spread technique.

Sterilize the loop again. Replace the cap or Cotton plug and incubate and after that refrigerate the slants.

Culture.

Place the loop with the bacteria into the slant tube, all the way down to the bottom of the slant. There are 2 ways to inoculate the slant:

If your goal is to identify the type of growth pattern, then just bring the loop straight up the slant.

If your goal is to have a luxuriant culture, inoculate in a zig-zag pattern, starting at the bottom of the slant. This increases the surface area of the culture.