Indentifying Bacteria: Capsule Stain Test

Today, we prepared a capsule stain in order to determine if bacteria 'L' has a capsule. First, we created a stain with Nigrosin by placing a drop on a corner of the slide. Then, we dropped a dollop of our bacteria 'L'into the Nigrosin using an inoculating loop and the aseptic technique. After mixing the two together, we took a clean slide and held it at a 45 degree angle and spread the drop across the surface of the slide, creating a thin, even smear. After some air drying, we were reading to begin the capsule stain. Then we used Crystal Violet to cover the smear  for 1 minute and than rinsed the slide (although not for too long as we did not want to rinse away 'L'). The slide than recieved careful blotting with Bibulous paper. It was than viewed under a microscope at 400x with immersion oil (prevents refraction of light as explained in previous posts!).

Bacteria 'L' & my throat sample (environmental) was found not to have a capsule. There was no transparent matter surrounding the bacteria in a 'halo' as our lab manual refers to it.

Myself holding the spread slide (Nigrosin & 'L' mixture)

Until next lab!

Annie B

Gram-Staining Bacteria

Today, we took a gram-stain of our bacteria 'L' in order to figure out if it was gram-negitive, gram-positive or gram-variable. Unlike the stain we did before (a simple stain) we used not just Crystal Violet but had to use Iodine (Gram's Iodine to be exact) and Safranin. However before we could use all the fun colorful chemicals above we had to recreate a simple stain. As mentioned previously (earlier blog post) we smeared, air dried and than heat-fixed the bacteria onto a slide. We than stained the bacteria using the Crystal Violet by covering the stain for 20 to 30 seconds over a slide drying rack. Then rinsing the slide with distilled water, we used Gram's Iodine and covered the smear for 1 minute. Then using 95% EtOH we held the size at a 45 degree angle and ran the color off the slide (decolored the sample). After this we rinsed again with distilled water and used Safranin to coverthe slide for 1 minute. Once finished we blotted the slide dry wth Bibulous paper and viewed it under a microscope on 400x using immersion oil. Our bacteria turned out to be Gram-negitive! This is because it did take up the Safranin! The peptidoglycan layer in Gram-Negitive Bacteria is so small that it cannot keep hold of the Crystal Violet once EtOH is applied. We know that because of this thin layer of peptidoglycan, the new color Safranin can hold (which is why the results below show a pink/red bacteria smear! (If it were Gram-Positive it would've been purple!)

 

this was the results under 400x

Rinsing Off The Slide With Distilled Water

The results at a lower magnification (100x)

Hope You Enjoied!

Annie B

Motility & Colony Classification of 'L' & A Strep Test

As mentioned in last blog, during the last lab we stabbed the samples into the motility test tube and left it to incubate. After pulling the tube out and holding it up to the light under a magnifying glass, it is noticeable that there is a cloudiness spreading out of the line where we stabbed the bacteria with the inoculating needle. This is a positive test for a motile bacteria! This means that bacteria 'L' is motile! It grew at the entire length of the tube as well. Since there is anaerobic conditions at the bottom of the tube it is probable that our bacteria is able to survive in anareobic and aerobic conditions. However this is not proven, just an inclination; further tests will need to be elvaluated. Most likely since it grew consistantly through the tube we hypothesize that our bacteria is facultative. This means that the bacteria grows by utilizing oxygen but that it can also adapt to grow without the oxygen!

My throat sample (environmental) did not have a halo of cloudiness surrounding the needle stab. Therefore it is non-motile. It has been growing in the incubator therefore we know that it is aerobic. However we could not tell if it grew all the way down the motile test tube. Therefore we hypothesized that it is most likely obligate aerobic bacteria. This means it can only grow in the presence of oxygen.

After testing for motillity, we used our broth samples (from last lab) to make depression slides (so we could observe bacteria 'L' swimming under a microscope!).  To begin, we flicked the broth tubes with our fingers. This swirling of the broth tubes caused a cloudiness to appear and reawaken the bacteria that had settled. We than took a slip cover to cover the depression slide and applied Vasoline to the corners of it. This helps it stick to the slide so the slide does not move (thereby making it easier to observe through a microscope). Using a sterilized, inoculating loop we applied the aseptic techinique to transfer a drop of the bacteria broth to the cover slip. Then flipping the depression slide upside down we stuck it to the cover slip (using the vasoline as the adhesive).  The drop was now suspended over the bacteria!
Using a microscope on 400x (highest power) and using immersion oil, we could observe 'L' swimming! It was so awesome!

As we observed our bacteria, we also noted key aspects that will eventually help us determine the true name of 'L' after several more tests. So far from our class examination we have discovered that 'L' is a rod-shaped (bacillus) bacteria and also exhibits a red color.

Back to the previous lab, we did an experiment to determine if Dr. P had strep. As you can see in the picture, only partical lysing occured. The green residue is a dead giveaway for incomplete lysis. Penicillian did kill the bacteria (meaning he had throat bacteria on the agar medium plate) however since bacitracin did not kill the cells there was no Streptococcus pyogenes on the plate (or therefore in Dr. P's throat!). Since complete lysis did not occur it is safe to say that Dr. P tested negitive for strep!Now it was time to take another look at the T4 bacteriaphage we placed in the incubator last lab. We used the same bacteriophage in two different agar plates (each containing a different bacteria). The agar plate with Dr. P's initials (JAP) the initials were clear all the way to the bottom of the plate, indicating that a complete lysis had occured. The other agar plate had no lysis occur! This proves that bacteriphages only affect specific hosts! This is why certain diseases are transmittible to humans through certain viruses! It is also why animal viruses & bacterias affecting humans was such a huge deal. If it can be transmitted to humans from animals, it is twice as contagious.

Incomplete lysis by antibiotics on blood agar plate

T4 Bacteriophage affecting bacteria

Until next lab!
Annie B

Streaking Plates & Creating More Stains

Today in lab we took our environmental sample and our unknown sample and placed the bacterias in test tubes containing broth. We fulfilled this procedure using the aseptic technique. After correctly transferring the bacteria, we labeled the tubes and placed them in the incubator at 25 degrees Celsius for the next class.

Following our lab books instructions for 'Examination of Bacteria and Creating A Pure Culture' we than made a streak plate of our unknown bacteria sample ('L'). This is so we could more closely examine the unknown and to perform more tests on it!

We also did a simple stain of the unknown bacteria 'L' (the slant which we took out of refrigerator) in a Methylene Blue stain. We followed the exact stain procedure as the one in the last blog post. It had to sit on the slide for a good minute before we washed it off with distilled water. We did the exact same Methylene Blue stain on our Environmental sample (in this case my throat swab!).

Staining With Methylene Blue 

Isolating the Bacteria into An Agar Plate

Classmate Preparing To Take A Swab From The Professor's Throat

After we did slides and viewed both the environmental & unknown sample under microscopes. We also made a new isolation of the unknown & environmental sample in peteri dishes (with agar). We also placed both the unknown and the environmental into broth tubes by using the aseptic technique. This ensures no contamination as we sterilize the loop between each transfer. We also started testing the Motility of our environmental & unknown samples. We took an inoculating needle and sterilized it in the flame of a Bunsen burner. Once sterilized, we used the inoculating needle to transfer some bacteria into a semisolid agar (this means there is .4% agar rather than 1.5%) and let it incubate for 24 to 48 hours at 25°C.

A classmate, Monica, then took a swab of our Professor's throat. She used a tongue depressor to make sure his tongue stayed out of the way and simultaneously used a swab over his tonsils.  It was really cool! We than rubbed the swab sample over red blood cells and placed two antibiotics in it: a small dose of Bactratin and a small dose of Penicillin (they were placed only in two concentrated areas on opposite ends of the dish). If the bacteria is only killed by the penicillin it is a normal bacteria that all human's throats contain but if it is also killed by the Bactratin it is Streptococcus pyogenes, which means the professor has strep throat! We currently left it in the incubator to grow!

Than our Professor used a T4 Phage in a peteri dish over a bacteria. He used a micro pipette to drop .5ml in one section of the dish and 1.0ml in another section. He then spelled out his initials: JAP in the center of the dish. (Although it is invisible at this point because the bacteria has not grown yet.) We than placed it in the incubator! Come back in two days to see the results of the T4 & the Professor's throat sample!

Annie B

A Microscopic look at Simple Stains and Unknown Sample 'L'

After making the simple stain last week, this week we placed the slides under a microscope and took a look at the bacteria! Once placed under the compound light microscope, we set it on the highest power-400x. We also used immersion oil to properly see the bacteria. Immersion oil is important because it keeps the light from refracting so that all the light goes into the view through the diaphragm and the bacteria is clearer. In this case I looked at my throat swab, it was fabulous. The bacteria from my throat is circular (cocci) but it was not in chains so it is not strepo form!! It was really cool to look at and I enjoyed being able to see the shapes of bacteria that we learned about in class live in the lab.

We also took the unknown sample out of the incubator! It had grown into a lovely red culture! We placed it in the fridge for next class :)

Microscope & Immersion oil fun!

Recording what I saw

Until next class,
Anne B

Staining Bacteria (Preparing an Unknown Sample & a Smear)

The class took out our streaking plates and observed the individual colony of bacteria that had grown on each one. We compared them to the Master Bacteria sample and some people had to redo them if individual colonies did not grow. Luckily, my sample grew properly so i did not have to re-streak my plate.
We took microscope glass slides (each person did two) and placed a drop of distilled water on them. We then sterilized loops and took a swab of bacteria from our petri dishes and mixed them into the distilled water. After we swirled them onto the glass microscope slides we let them air dry. Once dry we ran them through the flame from a Bunsen Burner to heat-fix the slide (sterilize). We than stained the slides with Crystal Violet and Safranin. The Crystal Violet slide had to sit for 20 to 30 seconds and the Safranin sat for at least 1 minute. Afterwards we rinsed the excess stain off with distilled water and blotted the slide dry with bibulous paper.

We then were given an unknown sample of Bacteria by our professor: Dr. P. We were instructed to identify the unknown sample of bacteria. Our bacteria was labeled 'L.' Before any tests can be done on the bacteria, we first had to create a pure culture of 'L' by using the aseptic technique. First we sterilized the inoculating loop and the top of the test tube that contained 'L' through a flame from a Bunsen Burner. The inoculating loop was then used to carefully obtain a sample of 'L' and spread it along a slant  agar test tube. The loop and test original test tube top were then sanitized once again through the flame and we placed the bacteria in an incubator at 25 degrees Celsius.

The Master Bacteria Sample (left) With The Streak Plate (right)

About to Heat-Fix The Slide

Checking My Slide To See If I Stained It Correctly

The point of keeping the Master Bacteria Sample was so that if the Streaking Petri Dish did not grow individual colonies we could use the Master Sample to redo the Streak Dish. We learned that isolating bacteria on an agar plate surface creates bacterial colonies in a pure culture from a mixture of bacteria. We also  learned how to properly stain bacteria which is important because staining bacteria makes it easier to be seen more clearly through a microscope. We also learned the proper size, shape, and arrangement of bacteria in a fixed smear. My slide displayed circular bacteria that was a slight green tinge in the crystal violet stain.

Until Thursday!,

Annie B

Isolation of Bacteria (Pure Culture in a Streak Plate)

Today the class took the bacteria out of the fridge to see how much it had grown. We looked at them closer under a microscope as well. My bacteria that was isolated from my throat was white colonies and were raised off the agar plate like little half bumps. There were also a couple yellow colonies by the white colonies. This signifies at least two bacteria types on the plate. Since there is more than one type we needed to create a select one and create a pure culture of it. I chose to create a pure culture of the white bacteria since there was more of it. To create a pure culture we took a inoculating loop and sterilized it with a Bunsen burner. Than we took a swab of one type of bacteria from our petri dishes and created a streak plate. We split the plate into four quadrants and spread the bacteria in a clockwise manner until it was spread out in the petri dish in the four quadrants. The purpose of this was to isolate the bacteria and to make it a pure culture. Once the petri dish was properly spread we labeled it with the date, name and type of sample (environmental). It was than set in the incubator at 25 degrees Celsius.

Master Bacteria Sample (that we took bacteria from for the new streak plate)

The new sample should grow into individual colonies and thus isolate one type of bacteria for a pure culture. This is important because in one speck of bacteria on the plate there are actually millions of bacteria growing there. The spread plate technique also is useful because with each quadrant the bacteria sample becomes more spread out and more isolated.

A Look At The Bacteria Through A Microscope

Streaking The New Petri Dis

Separating The Petri Dish Into Quadrants

Dr. Joseph Sterilizing The Loop In Between Streaking

Hope You Enjoyed,

Annie B

Isolation Of Bacteria At FUS

Annie B's Throat Sample #1

Every student in our Medical Microbiology Course #217 collected a bacteria sample from the first floor of Cosmos & Damian at Franciscan University of Steubenville. Using cotton swabs, each student first dipped the swab in chicken broth and then swabbed an area where they thought interesting bacteria would accumulate. Some students chose bathroom door handles, some chose cell phones, others chose I.D. cards and one student even selected a mysterious boy's bike seat. I chose to do my sample on my own throat bacteria. Since I was placing the swab inside of my mouth, which is already moist with saliva and therefore the bacteria would adhere to the swab without a problem, I did not dip the swab into the chicken broth beforehand. Dr. Pathakamuri helped me swab the inside of my throat and than I rubbed the swab thoroughly into an agar plate and closed the lib. I then labeled the sample with my name, what the sample contained and the date of the sample. The sample was than stored at 25 degree Celsius.

The Test Tubes of Chicken Broth

This lab is interesting because I do not often think of the places where bacterium resides. The study of microbiology is based on the Greek words 'micros' meaning 'small' & 'bios' meaning 'life'. (http://en.wikipedia.org/wiki/Microbiology). This is fitting because the small life is everywhere in our daily lives yet because it is so tiny we do not fully appreciate the mystery of the microworld. This lab is also teaching me how to properly take a swab sample and to place it in a peteri dish and keep it as uncontaminated as possible. This procedure is important as nurses to take samples to test in the labs to test for bacteria that cause diseases such as to test for strep throat (caused by a baterium called streptococcus). This procedure is also necessary in order to begin to take a pure culture of bacteria and determine which bacterium actually causes which diseases and where in our daily world they are found!

Annie B Placing Bacteria From Swab Into A Peteri Dish

This lab was very enjoible and at the end we even took check swabs and looked at our check cells through a microscope!

Swabbing of The Throat



Cool Microbiology Websites I Found While Researching The Root Of The Word 'Microbiology':

Microbe World

The Microbe Zoo

Molecular Muse

and my personal favorite:

Microbe Kombat

Until My Next Bacteria Sighting,

Annie B

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