Bacterial microscopy

Microscopy of culture of moss collected 7 days ago

Posted by Andrew Thornett

This sample was collected from our garden 7 days ago and kept in an open jar of water.

The contents of the jar had separated into a top layer of moss floating on the top, an intermediate layer of very cloudy water and a bottom layer of debris on the bottom of the jar. I have tried to sample all three layers in the pictures below.

Andy

x20 objective bright field sample from bottom of jar – debris layer. This shows large numbers of bacteria.

x32 objective bright field bottom debris layer:

Moss 7 day culture bottom jar layer video x32 objective Phase I annulus:

 

x20 phase contrast I debris layer bottom jar:

x32 objective phase contrast I debris layer jar:

x20 objective phase contrast I cloudy liquid layer between debris on bottom and floating moss – I am not convinced that this is phase contrast even though I labelled it as such – looks like bright field to me now:

x20 bright field liquid layer between debris and moss – video:

 

x20 bright field one single moss plant from the floating moss on top of the jar. If you look carefully you can see hundreds of bacteria surrounding this plant:

 

Microscopy of 2 week Petri dish culture of bacteria from hair in my armpit

Posted by Andrew Thornett

This may seem a weird experiment to have done – but 2 weeks ago Ean Ean snipped off some hair from my armpit directly into a Petri dish to see what we grew. The reason for this behaviour was that my T-shirts and jumpers tend to develop a lot of holes in the armpit areas and we wondered what bacteria was doing the damage. I do have reasonably hairy armpits!

Pictures below of the bacteria we grew – they are small round bacteria – this is known as coccus (cocci in pleural).

The Gram staining (by Rhys today) shows that the bacteria are Gram Positive.

A brief internet search shows that common axillary bacteria include Corynebacterium and Propionibacterium. Another bacteria that is often present but in smaller number is Staphylococcus.

Corynebacterium is a genus of bacteria that are gram-positive and aerobic. They are bacilli, and in some phases of life they are, more particularly, club-shaped, which inspired the genus name. The principal features of the Corynebacterium genus were described by Collins and Cummins in 1986. They are gram-positive, catalase-positive, non-spore-forming, non-motile, rod-shaped bacteria that are straight or slightly curved. Propionibacterium is a gram-positive, anaerobic, rod-shaped genus of bacteria named for their unique metabolism: They are able to synthesize propionic acid by using unusual transcarboxylase enzymes. Staphylococcus is a genus of Gram-positive bacteria. Under the microscope, they appear round, and form in grape-like clusters. The Staphylococcus genus includes at least 40 species (Wikipedia).

My bacteria appear to be Staphylococci – round and blue.

 

Preparing sample for microscopy today:

1. The Petri dish culture – axillary (armpit) hairs are visible!

2. The spatula was used to scrape off the bacterial culture into this container and then it was mixed with small amount water:

3. Small drop put onto a slide:

4. This is then dried:

 

Unstained heat-fixed slide, with 63x objective:

Gram stained slide, x32 objective:

 

Gram stained slide, x63 objective:

 

Live and stained microscopy (H&E and gram staining) of bacteria in single culture from previously taken mouth swab from Hannah 5/11/2017

Posted by Andrew Thornett

Today, was Hannah’s turn. Both children prepared slides from the culture we have grown from a swab taken a couple weeks back, stained with H&E and Gram stains and viewed live, H&E, and Gram stained samples under the microscope using x4, x20, x32 objectives. For some reason, we weren’t able to obtain focus with the 63x objective today – not sure why!

Our conclusion – Hannah has Gram positive rods in her mouth, most likely Actinomyces, possibly Actinmyces Israelii.

Actinomyces israelii is a species of Gram-positive, strict anaerobic, rod-shaped bacteria. It is known to live commensally on and within humans in the mouth, vagina and colon, A. israelii is an opportunistic pathogen. It was named after the German Surgeon, James Adolf Israel (1848–1926), who studied the organism for the first time in 1878 (Wikipedia).

Andy, Rhys and Hannah

Rhys and Hannah made these slides:

Microscopy-culture-form-Hannahs-mouth-051117-x4-obj-live-sample (below):

Microscopy culture form Hannahs mouth 051117 x20 obj live sample Ph1 (Phase contrast gave much better view than non-phase live images, although bacteria could be seen in both – in Phase contrast the bacteria appear dark as if stained (below):

Microscopy-culture-form-Hannahs-mouth-051117-x32-obj-live-sample-Ph1 (below):

Microscopy-culture-form-Hannahs-mouth-051117-x32-obj-Gram stain (the first one also has Phase annulus 1 in place, 2nd photo does not. In theory phase contrast should not be used on these samples but we found today it did enhance the image by darkening the background and improving contrast). It is clear that this bacterium is Gram positive (blue) and consists of rods:

Microscopy-culture-form-Hannahs-mouth-051117-x32-obj-H&E stain – again first image also has Phase annulus 1 in place and next two do not. Although the pictures look similar, the stains are not staining the same things. H&E stains nuclei and membranes blue and cytoplasm pink regardless of Gram staining characteristics. Some bacteria are Gram positive (blue) others are Gram negative (pink):

 

Live and stained microscopy (H&E and gram staining) of bacteria in single culture from previously taken mouth swab from Rhys 4/11/2017 & use of new SEP heater for heat-fixing and drying slides

Posted by Andrew Thornett

In my previous posts, I explained how the children and I took a swab of Rhys’ mouth and cultured it. Then, we took sample of one of bacteria that grew on the first culture to grow a second culture of a single organism. Today, I created a number of slides from this second Petri dish and then viewed them using 20x, 32x, 40x and 63x objective (equivalent when you add in the magnifying effect of the Bresser MikrOkular camera to up to 4300x magnification (see next post – 63x objective = 4300x magnification on screen) with live culture, H&E staining and Gram staining.

This represents my most successful observations of bacteria under a microscope so far.

The appearance and staining properties of the bacteria show that they are gram positive rods. Gram positive rods and filaments are organisms that are commonly isolated from the biofilms of dental plaque. The following organisms are commonly found:

  • Actinomycetes
  • Lactobacilli
  • Eubacteria
  • Propionibacteria

I am not sure which of these we have grown here!

Andy

Heat fixing slides for staining using new variable heater:

63x objective live sample (depending on size of the screen you are using to view this image this equates to around 4300x magnification), clearly showing strings of long bacteria:

Phase contrast (Ph I annulus) 63x objective:

H&E staining, 63x objective – with this stain purple represents nuclear material and cell membrane and pink the cytoplasm. As you can see, at this enormous magnification (4300x) sub-structural differentiation (i.e. clearly different parts – different areas are blue and pink) can be seen within the bacterial cells:

Gram stain, 63x objective – Gram positive bacteria are purple suggesting this is Gram positive bacteria:

Although the following image does not add any detail or information to those above, it does represent an increase in magnification. I used the Bresser Mikrocam 9.0 camera to photograph the H&E slide and then cropped a small section of the photograph and used the high definition in the image to allow me to magnify the image further. The above images did not involve any post-processing but the image below has been post-processed in GIMP2 using some sharpening, contrast enhancement and some enhancement of colour. As a result of this, the magnification has been increased on screen to roughly 8000x magnification!!:

H&E Staining of culture of Rhys’ mouth 29/10/2017

Posted by Andrew Thornett

I am very pleased with the following pictures – they represent some of best work with the microscope so far. They show good detail of bacteria. Heat-fixed onto the slide and H&E stained. The 63x objective images in particular are very effective – I am learning to use this objective now. Required the slide to be upside down to get focus.

I have converted images to greyscale.

Andy

Heat-fixing a slide:

H&E stained – 32x objective – following taken with Bresser MikOkular camera:

H&E stained images – 63x objective – Bresser MikrOkular camera:

H&E stained images – 63x objective – Bresser Mikrocam 9.0 camera:

Following picture is area of interest from above photo:

Fluorescein dye microscopy of culture of Rhys’ mouth swab 29/10/2017

Posted by Andrew Thornett

I tried again to use fluorescein 1% dye on a microscopy specimen today. I heat fixed a slide of the culture of Rhys’ mouth and soaked it in fluorescein dye for 10 minutes. I washed off the excess, added coverslip and looked at under the microscope. For this experiment, I used epi-illumination with the LED illuminator a second hand fluorescence filter block I obtained from ebay recently.

Pictures below – not sure what they show though!

Andy

Following is a non-stained live image of Rhys’ mouth culture through the Zeiss IM microscope with 63x objective as reminder of what is in the Petri dish culture used for this fluorescence experiment (this is NOT a fluorescence image):

Fluorescence image using fluorescein dye x4 objective (below). I am not sure what the darker blotches represent. This is a low magnification image so they do not represent individual bacteria but more likely large masses from the smear I took off the Petri dish using a needle. I had difficulty attaching the illuminator to the epi-illumination port today and it ended up slightly angled – hence the darker colouration to the left of this picture.

Fluorescence image using fluorescein dye x4 objective (below). The following two images look impressive but I suspect they are only of air bubbles!

Fluorescence image using fluorescein dye x20 objective (below). I have increased the magnification. The MikrOkular was able to show some detail, which is what was used for these photos. I also tried the Mikrocam but it was not sensitive enough. Again, I do not know what the darker patches represent. Far too low magnification for any features within bacterial cells (see size of individual cells in 63x image at start of this post):

Fluorescence image using fluorescein dye x32 objective (below). The highest magnification I could achieve and still see any detail. For this one, I had to stretch the image in GIMP2 and sharpen it. Again – what the features are I do not know. They vary in size from small ones, about the size of a bacterium to larger ones.

Hannah re-plates out Petri dish cultures from 22/10/2017 to obtain single organism cultures and adding penicillin to existing Petri dishes 29/10/2017

Posted by Andrew Thornett

Our Petri dish cultures of air born bacteria and fungi and mouth swabs from Rhys and Hannah from 22/10/2017 are now covering whole of Petri dishes. The air born cultures (open Petri dishes left outside for 4 hours to collect sample) show wide range of organisms – bacterial and fungal cultures – and the mouth swabs show much more limited number bacteria mainly (with smaller areas occupied by fungi). Interesting the swabs from the two children appear to have grown quite different bacteria, if the macroscopic view of the Petri dish is anything to go by. Rhys’ mouth swab has brown a gelatinous yellow-coloured shiny organism, whereas Hannah’s a rougher looking dry appearance to her culture.

Today, Hannah used a bent sterile needle on a diabetic syringe to attempt to plate out new cultures from the old ones on new Petri dishes – the idea is to obtain a single culture. She used her mouth swab culture Petri dish for this purpose.

After that, she poured some Benzylpenicillin powder (out of date penicillin from my surgery) into the old Petri dishes (her swab culture and one of air cultures) to see over this next week what effect that will have on the organisms present – she tried to get a straight line on the mouth swab – difficult to achieve as she did not want to touch the penicillin and contaminate it! – and more liberally splatter-gunned the air culture as this has lots different organisms and she wanted to see what the pencillin’s effect was on different ones.

As we do not know what we have grown here, there is a risk that some of these organisms are pathogenic (could lead to disease) so Hannah used sterile technique and gloves and we cleaned everything thoroughly afterwards!

Andy