Author: Deborah

Hi Started the day Gram staining E coli…

Deborah / / Uncategorized

Hi,
Started the day Gram staining E.coli a strain i dont have, this is another cultue confirmed so i then made a broth which i am incubating overnight at 37C. Spent most of the day waiting for cells to grow to an OD of 1, though 0.8 – 1.5 would be within range. Nicola went through serial dilution with me and showed me how to do the first set, i then completed the following three, broth culturing -4 dilution and spread plating -5 and -5.5 dilutions onto LB agar plates and incubated them overnight at 37C. Hopefully tomorrow we will have enough growth to count colonies (100 would be nice) then we can do replica plating. Had another Mike Shaw masterclass and debate (loving lab life xx).

Hi all Interesting day today the broths i…

Deborah / / Uncategorized

Hi all,
Interesting day today, the broths i innoculated friday were ready today so i made them into stabs and slopes. Finally my workspace is clearing, made up a few more bijou jars ready for the end of the week when i can hopefully add Enterobacter spp and Citrobacter spp to my completed list. I then went through the cell competency procedure with Nicola and she assaulted my brain with maths (hate maths mondays), we then went over plasmid stability and what we were to achive this week. in preparation we made up 400ml LB agar, 400ml LB agar with chl10, 200ml LB agar with Ampicillin 100 and 200ml LB agar with Ampicillin 50. Tomorrow serial dilution!

Evening all Started out the day by performing…

Deborah / / Uncategorized

Evening all,
Started out the day by performing an indole test, confirmed Kl oxytoca i have made a broth so that on monday i will make up stabs and slopes. Once the Kovacs reagent was added to Kl oxytoca a cherry red ring immediately formed on the top of the broth, meaning that it was a positive result and that Kl oxytoca has the ability to cleave indole from the amino acid Tryptophan. Also i have brothed up Lactobacillus and incubated at 25C so that it will be ready to make up into stabs and slopes on monday. My A.baumanni stabs and slopes are incubating at 37C until tomorrow. I have restreaked a couple of plates Ent. cloace, Enterbacter spp and Serratia marascens hopefully i will be able to confirm these next week. My work bench is clearing!!!

Awesome day in the lab today Made up…

Deborah / / Uncategorized

Awesome day in the lab today,
Made up a broth of 1g Tryptone and 0.5g Soduim chloride in 100ml sterilised water. this was then dispensed into universal jars (4ml per universal) and autoclaved at 121C for 15 minutes. Using aseptic techniques i the innoculated the broths with Kl terrigena, Kl pneumoniae and Kl oxytoca these were incubated over night at 30C. So hopefully they will have enough growth so i can add Kovacks reagent tomorrow (indole test) and i can hopefully differentiate the three culures. I have confirmed Listeria monocytogenes and so have made a broth and will hopefully make up stabs and slopes tomorrow. Alice has kindley donated a pure sample of A. baumanni, which i streaked onto a nutrient agar plate and also innoculated a nutrient broth and incubated at 30C overnight. I have also confirmed Streptomyces aureofaciens which i will broth culture tomorrow. Mike was amazing and explained loading dyes in detail, and how pH can alter substances, and how dyes can be used to range DNA banding during electrophresis (bromphenyl blue around 300bp). Then mike sprinkled a red powder (flourescens) into a beaker of water, because of the pH change the water began to glow like a yellow highlighter pen. Mike explained why this happened (outer shell valence and refraction of light) but i was happy with my glowing water xx Next Nicola explained how she was to cut out DNA from the agarose gel she had just ran and the purpose of this. Then i watched as she cut out each band of target DNA so that tomorrow the agarose gel can be dissolved away to leave behind the desired DNA. I Iooked at the kit to do this and it is in no way as simple as it seems. Even had time for an ethics debate with Dan, Sophie, Rosie and Kamilla.

Evening Firstly i checked on the Pseudomonas B263…

Deborah / / Uncategorized

Evening,
Firstly i checked on the Pseudomonas B263 that i had made into stabs and slopes, all are showing excellent growth, another culture cleared from my bench! next Sharon and i made a few slides using lactophenol cotton blue as we still need to practice our basic techniques. Then we Gram stained a few more of the bacterial cultures on my workbench, confirming as much as we can that they are what they should be. Because a few of my bacteria are in th same family and look the same under the microscope an indole test is required to confirm their identity.I have checked that we have everything required to carry out the test, so the job for morning is now to make up the broths ready for the test.

Hi all another quiet day in the lab…

Deborah / / Uncategorized

Hi all,
another quiet day in the lab but very interesting. First started by collecting water from the Brayford, then Sharon spread plated, MEA, Mannitol salt, Baird parker, MLSA and nutrient agar plates. Sharons project was to count colony numbers, now that is complete, we were curious as to the type of colonies. Next i innoculated a couple of MEA slopes with the three confirmed funghi on my work bench and made a couple of MEA broths using the two funghi i have as yet not been able to culture. Saccharomyces spp and Mucor you will not defeat me!!! Nicola had a meeting which clashed with the timing of her cells being ready for competency, so she talked me through the process. Really loved it, want to learn more about anti microbial resistance.

Hi all quiet day in the lab today…

Deborah / / Uncategorized

Hi all,
quiet day in the lab today. Made up 10X10ml universal bottles of MEA agar, as slopes so that tomorrow i can innoculate them with the confirmed funghi i have, Aspergillus paraciticus, Penicilin notartum and Aspergillus niger. Also made up 50ml of MEA broth so that i can hopefully encourage my remaining funghi to grow. Now to research Listeria

Hi today Luke spent a little time with…

Deborah / / Uncategorized

Hi,
today Luke spent a little time with Sophie and Dan, Big thanks guys x. Then we made up few slides using Lactophenol cotton blue to verify the fungal cultures i have. Penicillin notatum, Aspergllus niger and Parasiticus,these are now ready for me to innoculate into MEA slopes next week. The only two fungi i have not been able to culture are Sacchromyces and Mucor but thanks to Mike i know where to begin on monday.

Hi today luke did water filtration with water…

Deborah / / Uncategorized

Hi,
today luke did water filtration with water from the Brayford and innoculated nutrient agar plates with the filtered discs and incubated them at 30C for two days. The mannitol salt agar plates that had been spread plated with water from the Brayford showed large yellowy mucoid colonies, this was a positive for a Staphylococcus species. Using a sample from the mannitol salt plates and from the E.coli luke streaked the day before, Sharon and Luke performed Gram stains to compare the colonies. We then made up 1000ml of 50X TAE, and a 1% agarose gel for Steve. I have confirmd two more of my bacterial cultures, hopefully tomorrow i can inoculate them into broths and then into stabs and slopes.

Hi all firstly sharon luke and I collected…

Deborah / / Uncategorized

Hi all,
firstly sharon, luke and I collected a water sample from the Brayford, then we each spread plated a couple of nutrient agar plates with 0.1ml of the Brayford water and incubated them at 30C for two days.Then Luke streaked a couple of nutrient agar plates with E.coli and incubated them overnight at 37C. Luke spread plated a couple of mannitol salt agar plates with the Brayford water. We then watched Rosie innoculate her plasmids with antibiotics as she explained to us what she hoped to find. Explained water filtration to Luke as this is our task for tomorrow.

Hi all Interesting day started the day by…

Deborah / / Uncategorized

Hi all,
Interesting day, started the day by making 3000ml of TAE buffer. Next i ran a PCR test with a sample of my hair. When we looked at the gel under the UV some showed two bands and some showed just one band, either i’m a boy or luke is a girl. Think we may need to redo the test and look up which banding type (1 or 2) shows a male or female. Researched a few of the bacterial cultures on my bench, some are same species where an indole test is the only way to differentiate them.

Hi all Checked Lactobacillus plates Two do not…

Deborah / / Uncategorized

Hi all,
Checked Lactobacillus plates, Two do not look like the other three, after Gram staining a sample colony from all the plates, two had Gram positve small cocci. The other three showed long rod shaped gram negative chains, ths was more like what i expected to find. To be sure, i restreaked these onto M17agar with added Lactose and incubated them overnight at 37C. In preparation for tomorrows PCR tests i made up the solutions needed, working out quantities of HCl and Tris-HCl in 200mM. Microbiology is fun but maths makes my head hurt!!! big thanks to nicola for her little equation, i now can work molar solutions out but i think more practice is definately needed

Evening all x Yesterday did a PCR test…

Deborah / / Uncategorized

Evening all x
Yesterday did a PCR test with my cheek cells, mouthwash was yuck! but good news is i’m human. Today was a day of filling in COSHH and risk assessment forms, definately NOT a fun part of my day, but necessary. Confirmed M.luteus so made up stabs and slopes, Chromobacterium CV026 also confirmed, made into stabs and slopes. Made up a batch of TAE and another litre of 50x (Tris base, glacial aceitic acid, EDTA and made up to 1L with distilled water). The main thing i learned, is to not go to lunch and leave the tris to dissolve. increased pressure made it near impossible to remove the stopper, fortunately we are scientists – heating the glass whilst running cold water on the lid, hey presto, the stopper comes off.

Quiet day in the lab today A few…

Deborah / / Uncategorized

Quiet day in the lab today!
A few of the yeast cultures i had did not grow after their first innoculation onto MEA plates. I have re-streaked Mucor, Aspergillus niger and Saccromyces spp onto fresh MEA plates and incubated them at 30C. Penicillin notatum and Aspergillus parasitucus successfully grew and i have re-streaked them onto fresh MEA plates and incubated them for two days at 30C, from this i hope to gain a pure strain of the target funghi. Confirmed Chromobacteruim CV026 and have made a broth culture so that i can make up long term storage stabs and slopes. Also made up a batch of TAE with Rosie and Fran. Now to research exactly what it is and does.

Hi all Restreaked a few nutrient agar plates…

Deborah / / Uncategorized

Hi all,
Restreaked a few nutrient agar plates, hoping to find pure colonies of the target bacterial cultures that were incubating. One of the cultures didnt need to be streaked, as it was a confirmed bacteria, chromobacterium violeceum. Confirmed as it produces low, convex colonies with a dark purple/metallic sheen (violacein production). This culture, using aseptic techniques, was innoculated into nutrient broth and incubated at 30C for a coulpe of days. Then it will be made into stabs and slopes for long term storage. Researching more on selective agars to confirm more of the cultures on my lab bench. Made up more nutrient agar plates, counted colonies from Sharons Brayford pool project, cleaned lab – quite theraputic. Can’t wait to see what tomorrow brings.

Busy day today Started the day going through…

Deborah / / Uncategorized

Busy day today,
Started the day going through wednesday and thursday’s results with Rachael, so she knows where we are with our cultures, and so we can plan what we’re doing next. Taught Hammad how to do water filtration, made up 950ml of M17agar base with 10g Lactose suppliment (suspended in 100ml distilled water), both were autolaved before mixing. After lunch Rachael poured the plates. Made up 2800ml (7 x 400ml) of LB agar and poured plates with five of these. The other two were left for Nicola to add ampicillin and then pour the plates.I also made up 250ml of 0.85% w/v NaCl, this also needed to be autoclaved. Made up two stabs and slopes with the PS aeruginosa broth that i incubated overnight. Counted colony cultures from Sharon’s Brayfod water project.

A really successful day Yesterday i streaked 4…

Deborah / / Uncategorized

A really successful day!
Yesterday i streaked 4 Pseudomonas CFC agar plates with cultures of PS aeruginosa.Today the plates showed excellent growth and the cultures glowed florescent green. Gram staining showed that the cultures were Gram negative and rod shaped. All this was evidence that i had a pure strain of PS aeruginosa, I innoculated nutrient broth and incubated it overnight at 37C. Tomorrow i will make up two stabs and two slopes. One confirmed bacterial culture down twenty seven more to go!!! Using the Qcapture pro (microscope attached to a computor) i saved images of my Gram stained cultures to usb, hopefully i will post pictures soon.

Today I have seen some really good growth…

Deborah / / Uncategorized

Today, I have seen some really good growth on my streaked agar plates. Pseudomonas is growing nicely on CFC agar, and i am going to make up some agar to selectively grow Lactobacillus. Tomorrows task is to Gram stain the agar plates that have good single colony growth. My friend google will help me to identify the Gram stained bacterial cultures.

Today Rachael and I started to isolate single…

Deborah / / Uncategorized

Today Rachael and I started to isolate single colonies from the agar plates we incubated yeserday and streak them onto fresh nutrient agar plates. These have been placed in the 37C incubator overnight. Hopefully if there is growth, we can start to Gram stain them, further research will help us to correctly identify them and make up more stabs and slopes. We also streaked a number of yeasts onto MEA and placed them into the 25C incubator. I learned that it is better to incubate some cultures at a lower temperature for longer than at a higher temperature as this could possibly kill some cultures, especially if the higher temperature is at the top end of their tolerance range. Whilst looking at the agar plates from yesterday the P.S. aeruginosa looked cloudy and smeary, very similar to B. cereus and not the expectation of P.S. aeruginosa. We Gram stained a couple of slides using P.S. aeruginosa they were Gram positive, our research showed that it should have been Gram negative, rod shaped. Because of this we are going to streak P.S aeruginosa on Pseudimonas CFC selective agar. Hopefully tomorrow we shall see some results. Thanks to Nicola and Mike i am learning to tag,italics and google chrome.

Quiet day in the lab today made nutrient…

Deborah / / Uncategorized

Quiet day in the lab today, made nutrient agar and MEA plates ready for tomorrow. Todays task was to try and streak plates with bacteria that looked dead. The dried bacterial sample were ‘wetted’ by adding a little Ringers solution and then streaked on nutrient agar (with the exception of the fungi, they were streaked on MEA). Once this was done, a little research was needed to find out the incubation periods for the target bacterial streak plates. On to more research to find out what each colony should look like and whether it is Gram positive or negative. Hopefully we can positively isolate and identify each bacterial culture and create stabs and slopes.