Week of 08/12/24 Twitch Motility Assay

 


Introduction

               Bacterial species lacking a flagella may still be able to move by dragging themselves across a surface using pili. This form of motion, referred to as twitch motility can be observed on thin media plates over a long period of time.

 

Methods

               Skim Milk Agar 10ml plates were utilized to observe twitch motility because the thin translucent media is conducive to observing the spread of bacteria. 1.5% skim milk plates were poured with a 0.75% agar concentration. Eight plates were poured – two plates for each of the following species: Deinococcus xinjiangensis, Deinococcus radiodurans, Deinococcus ficus, and Deinococcus aquaticus P81. Each of the species were gram stained from a stock plate and inoculated onto the skim agar plates using a specialized method. A sterilized bore was first used to remove a cylinder of media from the center of plate. A sterilized toothpick was then used to pluck a single colony from the stock plate and to place the colony into the center of the hole bored onto the skim milk plates. It is important that the colony is placed directly onto the plastic of the petri dish to minimize the number of bacteria that will grow up the side of the cylinder of media up onto the surface of the plate. Twitch motion is observed specifically along the bottom of the petri dish on the underside of the skim milk agar.

               The plates are then observed and documented to observe the spread of bacteria over time. Two phenomena are observed: the enzymatic breakdown of proteins and sugars and the “feathering” of bacteria that indicates twitch motility.

 

SMA (1.5% SM with 0.75% Agar

               250 ml H20

               3.75 g Skim milk powder

               1.875g agar

SMA (0.75% SM with 0.5% agar)

               100 ml H20

               0.75g Skim milk powder

               0.5 g agar

TGY broth (1000 ml H20)

               3g yeast

               3g tryptone

               1g dextrose

TGYA (1000 ml H20)

               3g yeast

               3g tryptone

               1g dextrose

               15g agar

TGYA with Chloramphenicol (3 micrograms per milliliter)

               200ml TGYA

               17.6 microliters of 34000 microgram per milliliter chloramphenicol stock

LB broth 1000 ml H20

               5g yeast

               10g tryptone

10g NaCl

LBA 1000 ml H20

               5g yeast

               10g tryptone

               10g NaCL

               12g agar

 

LB Amp at 50 micrograms per ml

               40 microliters AMP

               20ml LB broth

 

SMA sigma aldritch 1000ml H20

               5g casein

               2.5g yeast

               1g skim milk powder

               1g dextrose

               10.5g agar

TGYA at 0.1x 1000ml H20 and 0.5% agar

               0.3 g yeast

               0.3g tryptone

               0.1g dextrose

               5g agar

TGYA at 0.5x 1000ml H20 and 0.75% agar

               1.5g yeast

               1.5g tryptone

               0.5g dextrose

               7.5 g agar

  

Results

               The preliminary plates showed different results for each species.

               The ficus plates had a medium amount of twitching motion and a medium amount of enzymatic activity. The xinjiangensis plates had a larger amount of twitching and enzymatic activity. The radiodurans plates had very little twitch and the largest amount of enzymatic activity. The aquaticus plates had very little enzymatic activity and a vast amount of twitching motility.

               The preliminary design of the plates led to cracks in the agar from the bore. These cracks would spread into fissures as the plates dried out.

 

Discussion

               The preliminary results suggest that aquaticus has the greatest amount of twitch while radiodurans has little to no twitch. Yet, aquaticus has the least amount of enzymatic activity while radiodurans has the greatest amount. This raises the question of whether or not enzymatic activity and twitching motility are inversely proportional, perhaps due to a limited amount of energy and resource allocation.

               The plates are going to fissure if the incision points continue to have tears in them. Thus, a new design is needed.

 

Comments

Post a Comment

Popular posts from this blog

Week of 11/25/24

Image
  Introduction The use of proteinase K has been inconclusive thus far and so we have decided to increase the concentration to 20ug/ml. Proteinase K is optimized at 50-65 degrees Celsius so its activity is slowed at the incubation temperature of P81. Hopefully increasing the concentration will account for the decrease in temperature.  Additionally, lab is closing soon for winter break and winding down protocols are in effect. Media production has been halted to as needed only and freeze backs of samples are being created.  Methods Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stain Plates and Broths after at least 24 hours 5) Record and compare results 6) If needed, introduce a protease to break apart pili connections 7) Re gram stain after 24 hours 8) If needed, run an RNA sequence to check gene express...
Read more

Week of 11/18/24

Image
  Introduction The plans for twitch and morph were put into effect this week. We studied the effects of proteases upon the connected P81 cells while simultaneously studying the effects of chemoattracts upon twitching P81 cells.  Methods Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stain Plates and Broths after at least 24 hours 5) Record and compare results 6) If needed, introduce a protease to break apart pili connections 7) Re gram stain after 24 hours 8) If needed, run an RNA sequence to check gene expression Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA and 0.5% agar) 2) Prepare a sterile hood by wiping everything down with ethanol 3) Melt media down 4) Wipe down all inoculating materials and insert them into the sterile hood 5) Dispense 10 ml of media into empty petri dishes co...
Read more

Week of 09/16/24

Image
  Introduction The transformation procedure was attempted again this week with a particular interest upon the OD value. We are attempting to perfect the procedure in every conceivable way so that any failure for P81 to transform can not be attributed to human error. Additionally, varying amounts of plasmid were used across experimental groups to test whether plasmid concentration has any effect. Methods A 278 ng per microliter plasmid extraction was used. The first experimental group was given 4 microliters while the second experimental group was given 8 microliters. The final experimental group was given 12 microliters of a 220.4 ng per microliter plasmid extraction.  Transformation Protocol 1) 1 ml cells OD value of 1 (late log) 2) Pellet 3) Resuspend in 100 microliters of TGY 4) Add 40 microliters of 0.3 CaCl2 mix 5) Ice 15 minutes 6) 30 microliter sample and about 10 microliters of plasmid (where the plasmid is 200-400 ng per microliter for a grand total of...
Read more