IGL2 Identification of Gall causing pathogen by employing Koch Postulates NC State University Department of Microbiology Patrick Sheehan Abstract The purpose of this experiment was to isolate a pure culture of the suspected gall causing pathogen Agrobacterium tumefaciens by employing Robert Koch s Nobel Prize winning Koch s Postulate s The suspected pathogen was isolated from a previously infected plant Then used to form a purified culture which was then used to infect a healthy subject The test subject plat then successfully formed a gall at the site of infection This gall was then surgically removed and tested for the presence of Agrobacterium tumefaciens After a positive confirmation of the suspected pathogen was made a second pure culture was then formed and used to be matched with the original pure culture This determinate match was made through a series of biochemical analysis tests It was then determined that both cultures contained the same species Agrobacterium tumefaciens and that the suspected pathogen did indeed cause crown gall disease in sunflowers By employing the same methods that Robert Koch formed in 1884 the experiment successfully proved Agrobacterium tumefaciens was responsible for crown gall disease in the tested sunflowers Intro In 1884 Robert Koch developed an algorithm for successfully identifying specific microbes that cause particular diseases The Koch postulates There are 4 separate criteria that need to be followed in order to accurately carry out the Koch postulate they are as follows 1 The suspected bacteria has be identified in all test subjects and may not be present in healthy subjects 2 The suspected bacteria needs to be isolated in a pure culture entirely from a diseased subject 3 A sample of the pure culture must then be used to inoculate a healthy host subject 4 The same pathogen must then be re isolated from the previously infected host and then compared with the original pure culture to get a positive match These are the four criteria of the Koch s Postulates This method of identifying pathogens went on to win the Nobel Prize in medicine in 1905 and has since then been used to successfully identify thousands of pathogens Agrobacterium tumefaciens is the known bacteria pathogen responsible for grown gall disease in plants This now infamous bacteria employs the use of plasmids to alter the genetics of infected organisms Van Larebeke 1974 However recent advances in biotechnology have allowed for the ability to insert useful genes into target organisms In face Agrobacterium tumerfaceisns plasmids have been used for a variety of industrial applications such as improving crop yields in rice Hiei et al 1997 In this experiment it is being tested to confirm that the Agrobacterium tumerfaceins is indeed the causative agent for grown gall disease in sunflowers Methods A sunflower was planted in loam soil half an inch deep inside a Styrofoam cup and allowed 4 5 weeks to successfully grow into a sapling In the meantime a gall from a previously infected sunflower was taken and removed from the plant The gall was taken and placed in a sterile petri plate This gall was then crushed via a hammer and soaked in PBS solution for 40 minutes From her a 1mL sample of liquid from the petri dish was taken and used to perform 6 tenfold serial dilutions These serial dilutions were then taken and plated onto 3 separate agar plates R2A differential OGYE selective and TSA non selective All 6 plates of each agar were then inoculated with 100uL of each level of dilution After each was plate was inoculated they were incubated for 7 days to allow for the development of biomass At the same time a sample of healthy plant sampling was taken and used to perform serial dilution of the same folds and amounts This healthy plants dilutions were then taken and plated on the same three agar plates and also incubated for 7 days to allow for biomass to form After 7 days had passed a sufficient biomass had been formed of both the gall plates and the healthy plates Colonies from all 3 plates from both the gall and healthy dilutions were compared to help establish suspect CFU s of Agrobacterium tumefaciens A distinct CFU was selected from plates with countable number of colonies from the gall dilution plates The CFU was then gramstained and sub cultured onto provided media plates for further biochemical testing The subcultures were plated on LB and LB Rif plates and given one week s incubation period to allow for the generation of biomass to be used in further testing The sub cultured CFU s then underwent a variety of field tests The test included mannitol utilization using Bromthymol blue mannitol medium urease production test using pheol red urea medium motility test extracellular protease activity test using milk agar medium cellulose 4 synthesis test using cellulose detecting medium citrate and utilization using ferric NH ammonium citrate medium and nitrate reduction was indirectly tested via selenite medium The Bromthymol blue mannitol phenol red urea motility and ferric ammonium testes were done by 4 inoculating a subculture onto agar in tubes While the Motility and citrate tests were NH inoculated by stabbing into a liquid solution The remaining tests were done in petri dishes All test done with both the gall and control isolates All test were then allowed one week s time to be incubated and develop biomass At this point in the experiment the sunflower plants that were originally planted had grown into saplings These saplings were then inoculated using the gall and control cultures from cellulose detection medium A small puncture was made in two distinct separate locations on the healthy sunflower plant and each of the punctures was then infected with either the gall or control isolates The plants were then left for three weeks to allow for the formation of a gall to take place After recording the results of infection the suspected gall causing microbe was to be reisolated from the diseased host tissue in this case the newly formed gall The newly formed gall was then surgically removed from the host plant and placed into a petri dish This gall was then crushed via hammer in the same manner as the original gall The biomass of the crushed gall was then soaked in 2 mL of PBS for 40 minutes At the conclusion of this timeframe a 100uL sample was taken and put into solution This solution was then used to form a T Streak onto the designated selective medium The plate was then incubated