JASMIN, KISHA JANE P.

INT 4218

On the first part of the video titled “Inoculation”, includes the inoculating loop sterilization which
is an important step in performing bacterial culture for this will prevent the contamination of the
growth of the bacteria which might cause interference in the diagnostic reading of results. Also
included in the video is the correct processing of respiratory specimen which shows the proper
streaking on the agar medium which yields a desirable growth of the bacteria of interest. Lastly,
a video which tackles the step-by-step procedure on the processing of urine specimen was
shown. This is equally important as with the two latter videos mentioned and are considered to
be the basic knowledge one must possess upon entering the Bacteriology laboratory.

The second part of the video uploaded with the title “Inoculation methods”, they discussed the
correct procedure on how to inoculate from one plate to another. First thing to do is to sterilize
the loop until it is already glowing red, let the loop cool because this will kill the bacteria. Lift the
lid and identify an isolate of colony then open the plate to which it will be transferred. The
correct streaking must be performed. The MacConkey agar is the second plate and this is
identified to be a Selective and Differential agar. Selective because only Gram negative bacteria
will grow on it, and it is Differential because you can tell the difference of growth according to
color as to whether it is Lactose-fermenter or not. The same procedure of inoculation was done
except that a different streaking pattern as done because of the need to isolate a colony of
bacteria. The bacteria being inoculated is Pseudomonas aeruginosa, a Gram negative organism
commonly found on the soil and causes various infection in humans. A colony is again
inoculated using a loop and transferred into an Simmons Citrate Agar slant observing correct
sterilization of the loop. The next tube is called the TSI (Triple Sugar Iron) slant, inoculate the
colony using the initial collection from the stock plate into the slant but the difference is that they
used a needle for inoculation where the colony is stabbed on to the bottom of the agar this is
because some organisms grow on the bottom and on the slant and that is what we are going to
observe/study.

The third video with the title “Inoculating Liquid Bacterial Culture”, discussed the correct
procedure of inoculation of liquid culture as it was described in the title. First, inoculation was
described as the process by which bacteria is introduced into a liquid medium. In this video,
they demonstrated best practices to perform inoculation ensuring sufficient numbers of bacteria
for plasma DNA isolation. By starting with a small liquid culture from a single colony, you can
ensure that the liquid culture contains a monoclonal population of cells. The materials needed
include: Spray bottle with 70% isopropanol, pre-made liquid LB media, LB agar plate containing
colony of plasmid of interest, 15 mL conical tubes, antibiotic, Bunsen/alcohol burner, autoclaved
toothpicks, shaking incubator, 10 mL serological pipette and pipette aide. First step is to
decontaminate the bench with 70% isopropanol and then prepare the materials needed for
inoculation. First, you will need to prepare you liquid growth medium. LB broth is usually used
as it is the most widely used medium for bacterial culture. If your media does not contain
antibiotic yet, you will need to add the appropriate antibiotic before proceeding. For any plasmid
requested for “addgene”, the antibiotic resistance will be listed on the Plasmid page under the
“Growth in Bacteria” section. Our plasmid contains ampicillin resistance. At addgene, their lab
uses carbenicillin instead of ampicillin. Carbenicillin is more stable than ampicillin. If your lab
uses ampicillin, try not to use plates that are more than a month old. Always make sure to look
at the correct antibiotic concentration needed for plates and media which can be found in the
table of addgene website. Since we’ll need 10 mL of liquid media for our inoculations, transfer
10 mL of LB medium into a 15 mL conical tube. Next would be the addition of antibiotics. 10 uL
of our 1000x Carbenicillin stock goes into our 10 mL of LB media. Add the appropriate amount
of antibiotic on the liquid media, making sure that the pipette tip does not touch the neck of the
bottle, reflame the tube, re-cap the lid and invert the liquid medium to mix the antibiotic. The
next step would be to arrange three culture tubes in the rack. Label the first tube as the
“Negative control” and label the other two tubes with the name of the plasmid. It is always a
good idea to select more than one colony for inoculations. To track these samples, we will label
“Plasmid A” and “Plasmid B” indicating same plasmid but different colonies. It is also a good
idea to add initials and the date on the tubes. Your negative control tube provides quality
assurance. This tube will contain no additional bacteria. Any growth in your control tube
indicates a contaminated culture. Transfer 2 mL of the LB Carbenicillin media into each culture
tubve using a serological pipette.Using a sterile toothpick, select a single colony from your LB
agar plate, remove the culture tube cap and drop the toothpick on the culture tube and label
your plasmid name and replace the cap, make sure the toothpick does not touch the outside of
the tube. For the negative control tube, simply drop the toothpick on the sterile tube. You can
use sterile caps or aluminum foil to cover your tubes. Ensure that the cover is not airtight, since
bacteria need air to grow. Most bacteria should be incubated at 37 degrees Celsius. Check to
verify the proper incubation temperature for your bacterial stain. Bacterial liquid culture should
be incubated on a gentle shaker to ensure proper nutrient availability. Incubation using a shaker
also avoid bacterial clamping at the bottom of the tubes. You can incubate your cultures in a
non-shaking incubator. If you do, you will need to incubate the cultures for one day or longer to
ensure an adequate number of cells. If you have a low copy number plasmid, you may need to
grow your culture longer than overnight. To determine if your addgene plasmid is high or low
copy, see the “Copy number” under the “Growth in Bacteria” section. After incubation, check all
tubes for growth. Your negative control tube should appear clear and no bacterial growth. So
what happens if after growing you culture overnight, you do not observe any bacterial growth?
First, allow the culture to grow more time as some bacterial cultures have slower growth rates.
Bacteria incubated at 30 degrees Celsius rather than 37 degrees Celsius often require longer
incubation times. Double check that the antibiotic in liquid media matches the antibiotic
resistance for your plasmid. Using the wrong antibiotic will yield no bacterial growth. If the single
colony used for inoculation was not freshly-streaked, you should re-streak your bacteria on to a
new LB agar plate before growing in liquid culture. Fresh colonies usually yield greater bacterial
growth. Normally, cultures shake at 150-250 rpm. Try increasing the speed from 350 to 400 rpm
to achieve greater cell density. Once you obtained adequate culture growth, you can spin down
your liquid cultures and isolate your plasmid DNA by following the DNA isolation protocol. For
long term storage, proceed by creating a Glycerol stock.