Rhys, Hannah and I have just spent a couple of interesting hours preparing and viewing slides from petri-dish cultures that have been developing (much to Ean Ean’s disguist) on the window ledge in the spare bedroom for the last 2 weeks. One was from a finger print Hannah made in the petri dish and the other from a swab of Rhys’s mouth from around the teeth (in an attempt to get some plaque).
…..The children prepared the slides under my supervision using aseptic technique (very important – human samples – need to know what you are doing as serious risk of pathogenic bacteria and serious illness – don’t do it unless you are properly trained).
We looked at these slides using the Zeiss IM microscope and 3 objectives:
- 4x objective – brightfield and with Phase 2 annulus (combination works really well to give excellent dark background so not sure whether this is really phase of simply darkfield but either way the view is great)
- 20x objective – brightfield and phase contrast
- 32x objective – brightfield and phase contrast
These latter 2 objectives were designed for this microscope and work incredibly well – when viewing samples their views have excellent depth of focus , long distance working, effective work with existing phase annuli and give contrasty bright views especially with my www.retrodiode.com 20W illuminator.
We prepared 7 different petri dish cultures initially and chose two today from which to prepare slides (below):
The following two photos show that, after two weeks, Hannah’s finger mark had led to growth of milky white bacterial culture, 2 white fungal areas, a yellow organism at around 2 o’clock and number of areas with pale haloes suggesting the production of antibiotics (below):
Hannah is taking a sample of one of the white fungal areas to put on a slide using homemade loop (below):
Rhys is looking at his mouth swab sample, as he gets ready to prepare his slides (below):
Hannah-fingerprint-in-petri-dish-white-fungus-culture-x4-objective-080717.bmp (below). This shows thousands of tiny blue/black dots. The round circle at top right is an air bubble:
Hannah-fingerprint-in-petri-dish-white-fungus-culture-x40-objective-080717.bmp (below). The extra magnification shows strings of spores in strings. No bacteria were present:
Hannah-fingerprint-in-petri-dish-bacterial-culture-x4-objective-080717.bmp (below). Again, at this low magnification, thousands of tiny dots seen but these turned out to be quite different from those seen above – this was on a different part of the slide – the milky white stuff that occupied most of the Petri dish:
Hannah-fingerprint-in-petri-dish-bacterial-culture-x20-objective-080717.bmp (below). With 20x objective (200-400x magnification), the dots resolve into strings of 1-3 cells:
Hannah-fingerprint-in-petri-dish-bacterial-culture-x32-objective-brightfield-080717.bmp (below). With 32x objective, green and blue/black organisms can be distinguished from each other in brightfield illumination:
Hannah-fingerprint-in-petri-dish-bacterial-culture-x32-objective-phase-I-080717.bmp (below). Phase contrast brings out the cells more effectively and demonstrates thousands of organisms, many in strings of cells but other isolated round organisms also present. Some are green indicating photosynthesis is occurring within them but others are not:
Hannah-fingerprint-in-petri-dish-bacterial-culture-x40-objective-080717II.bmp (brightfield, below). The following two images demonstrate that the 40x objective does not add much to the 32x objective data. In fact, the 40x objective here has smaller depth of field and this is a problem in terms of focusing on the organims in this unfixed specimen:
Hannah-fingerprint-in-petri-dish-bacterial-culture-x40-objective-080717.bmp (bright field, below):
Rhys-swab-around-teeth-petri-dish-culture-x4-objective-brightfield-080717.bmp (below). Again, a debris field with thousands of blue/black dots:
Rhys-swab-around-teeth-petri-dish-culture-x20-objective-brightfield-080717I.bmp (below). With magnification, the dots resolve into bacteria:
Rhys-swab-around-teeth-petri-dish-culture-x32-objective-brightfield-080717I.bmp (below). Now it is clear that there are some long organisms which might be actinomyces, believed to be involved in dental decay and uggesting Rhys needs to make mlre dffort to clean his teeth, and other small round blue/black organisms present, possibly Streptococcus mutans, a common commensal in the mouth:
Rhys-swab-around-teeth-petri-dish-culture-x32-objective-phase-I-080717I.bmp (below). Phase contrast brings out the fact that many of these organisms are in strings:
Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x4-objective-phase-II-080717I.bmp (below). Rhys was interested in what was growing inside one of the areas with a clear halo around it. The clear halo implies that a bacteriocidal substance is being produced. The image below shows that there are large numbers of organisms of different types in the centre of these halos with many interesting structures with a boundary and lots of organisms of one particular type within, as in the bottom left of the following image:
Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x4-objective-phase-II-080717II.bmp (below). This gives a view of one of these structures (left of image):
Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x20-objective-brightfield-080717I.bmp (below). Outside of the boundaried structures mentioned above, there are bacteria but, unlike the initial views within the bacterial area that Rhys first sampled, these bacteria are largely not moving – occasional small round fast bacteria moved through the field of view but otherwise these bacteria appeared dead:
Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x32-objective-Phase-I-080717I.bmp (below). Within one of the boundaried structures, the cells form lined structures several cells long and sometimes branching. They appear to all be the same. The boundary is not composed of cells but its two colours suggest it is the boundary of a matrix of one refractive index with another. To me this suggests that the cells in the middle modify the fluid in which they live.
Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x32-objective-Phase-I-080717II.bmp (below). This clearly shows the boundary between the boundaried area (left) and bacterial area (right). Note both of these are within the clear halo (milky white bit in the middle of the halo on the Petri dish) – I suspect that the organisms on the left within the boundary are fungi and are producing an antibiotic that has killed the bacteria both within and outside of the boundaried area. The limit of its effectiveness is up to the outer edge of the halo after which we see bacterial growth again: