RESULTS
Sampling of the surface contamination using sterile cotton swabs from toys obtained from
the Nursery at Queen Mary, University of London showed diverse microorganism growth.
A variety of species were cultured and identified successfully from serial dilutions that
initially took place in order to produce solutions of low molar concentrations that could then
be plated. The wooden toy inoculum was diluted to 10-5 and three distinct colony types,
designated WA, WB and WC, were shown to have grown when cultured on nutrient agar
plates, spread using a bent glass rod. Inoculum from the soft toy was diluted to 10-4 and two
distinct colonies of microorganisms, designated SA and SB, showed very visible, strong
growth when cultured on the nutrient agar. Finally, inoculum from the plastic toy was
diluted to 10-4 and showed strong growth of only one type of colony, designated P.
Following procedures as mentioned in the "Methods and Materials" section, firm
conclusions were drawn as to the identity of the bacterial species and the distinct colonies
on each of the toys.
Below are the tables indicating the results from each of the tests undertaken:
Table 1. – Identified bacteria from each toy and their distinct colonies
Wooden toy (10-5) bacterial
growth
Colonies WA – Mycobacterium phlei
WB – Staphylococcus aureus
WC – Bacillus cereus
Soft toy (10-4) bacteria growth
SA – Staphylococcus epidermidis
SB – Bacillus cereus
~ 21 ~
Plastic toy (10-4)
bacterial growth
P - Bacillus subtilis
Table 2. – Results of Gram-staining procedure
Gram Stain
Gram positive organismsGram negative organisms
WB – observed as coccus
WC – observed as rods, possible spores
SA – observed as coccus
SB – observed as rods
P – observed as rods, clear sporulation
WA was inconclusive
WA – Mycobacterium phlei, WB – Staphylococcus aureus, WC – Bacillus cereus
SA – Staphylococcus epidermidis, SB – Bacillus cereus, P - Bacillus subtilis
Table 3. – Results of Catalase test
Catalase test
Catalase positive
WB
WC
SA
SB
P
Catalase negative
WA
WA – Mycobacterium phlei, WB – Staphylococcus aureus, WC – Bacillus cereus
SA – Staphylococcus epidermidis, SB – Bacillus cereus, P - Bacillus subtilis
~ 22 ~
Table 4. – Results of Dryspot Staphytect Plus for the identification of
Staphylococcus aureus
Dryspot Staphytect Plus
Positive result
WB – agglutination
Negative result
WC
SA
SB
P
WA
WA – Mycobacterium phlei, WB – Staphylococcus aureus, WC – Bacillus cereus
SA – Staphylococcus epidermidis, SB – Bacillus cereus, P - Bacillus subtilis
The visible agglutination implied that this unknown was likely to be Staphylococcus aureus.
No visible agglutination from SA (Gram-positive coccus) implied that the unknown was
possibly Staphylococcus epidermidis.
Table 5. – Results for the growth on Mannitol Salt agar to distinguish between
Staphylococcus.
Mannitol Salt agar
Growth
WB – pale yellow colonies observed
SA – pink/red colonies observed
No growth
WC
SB
P
WA
WA – Mycobacterium phlei, WB – Staphylococcus aureus, WC – Bacillus cereus
SA – Staphylococcus epidermidis, SB – Bacillus cereus, P - Bacillus subtilis
Growth on the highly selective mannitol salt agar led to confirmation of unknown WB as
Staphylococcus aureus.
Unknown SA was confirmed as Staphylococcus epidermidis due to characteristic growth.
~ 23 ~
Table 6. - Results for the growth on Bacillus cereus agar to distinguish between
Bacillus
Bacillus cereus selective agar
Positive resultNegative result
SB – pink colonies observedSA
WC – pink colonies observedWA
P – yellow colonies with slight precipitateWB
observed
WA – Mycobacterium phlei, WB – Staphylococcus aureus, WC – Bacillus cereus
SA – Staphylococcus epidermidis, SB – Bacillus cereus, P - Bacillus subtilis
Visible pink/red colonies confirmed the already suspected presence of Bacillus cereus
following catalase testing and also viewing the unknowns SB and WC from under the light
microscope.
Yellow colonies are characteristic of Bacillus subtilis.
Table 7. - Results of spore staining and acid fast staining to identify possible
Mycobacterium phlei
A spore stain was undertaken with unknown colony WA to distinguish it from possible
Bacillus cereus. Under the lens of the light microscope it was difficult to differentiate
whether the unknown was rod-shaped or cocci-shaped. Following a negative test result, the
acid-fast stain was then undertaken to determine whether the unknown was indeed
Mycobacterium.
Spore stain
Acid-fast stain
Unknown colony WA
Negative result
Positive result
~ 24 ~
As shown, Bacillus spp. were found to be common. Bacillus endospores are often found to
disperse rapidly through the air and in soils and other environments are an ever-present
microorganism (Nicholson, 2002) (Vilain et al,2006) so their abundant presence here was
not startling.
This is predominantly because the toys that are played with are often moved from the
inside and the outside of the nursery premises, by children who are running around.
Therefore, if dropped on the ground or more precisely in areas of grass and soil, the toys are
able to accumulate such bacteria and these bacteria can then be transferred onto other toys
as well as children from inside the nursery. In addition to this, Staphylococcus spp. were also
identified as well as Mycobacterium phlei.
Bacillus cereus is a prevalent, gram-positive, rod-shaped bacterium that resides in soil. Most
commonly, Bacillus cereus is known to be harmful to humans due to their ability to cause
illnesses arising from improper preparation of food. Cases of food-borne illnesses
predominantly arise due to the production of bacterial endospores produced by the
microorganism and their survival (Turnbull, 1996). These endospores, and the general
bacterial growth, may then lead to the production of enterotoxins and the onset of nausea,
diarrhea and vomiting in humans (Kotiranta et al, 2000).
In the case of children within a healthcare environment, the transmission and possible onset
of disease is therefore of obvious concern. As acknowledged, the spores of B. cereus can be
found widely in the nature around us, including samples of dust, dirt and water amongst
others. Normal contamination levels are generally known to be <100/g (Hobbs and Gilbert,
1974) however such levels are unlikely to be found within a carefully and often sanitized
child-care environment.
~ 25 ~
Bacillus subtilis, a gram positive rod, is commonly found in plant undergrowth and soil as
well, as such, it is often known as the grass Bacillus (Madigan and Martinko, 2005). This
particular species of Bacillus is also known to be non-pathogenic in humans. Much like
Bacillus cereus, they are acknowledged to contaminate food products but are nowhere near
as potentially harmful as Bacillus cereus and very seldom result in food-borne illnesses prior
to ingestion of the contaminated foodstuffs (Ryan and Ray, 2004). Therefore, incidence of
the bacteria, particularly in a child care environment can be regarded as harmless.
Staphylococcus spp. were also cultured and identified. This is a genus of Gram-positive
coccus that are said to form in "grape-like" structures when viewed under a light
microscope following staining (Ryan and Ray, 2004). It is well understood that most
Staphylococcus are harmless and are commonly found to reside on the skin and mucous
membranes of humans very comfortably (Kluytmans et al, 1997). It is interesting to note
that Staphylococci are microorganisms that also universally dwell in soil and dirt.
Staphylococcus aureus in particular is a well known specie of the genus and resides in the
nose and on the skin of healthy human beings (Kluytmans et al, 1997). Research has shown
that seemingly almost 20% of the human populations are carriers of Staphylococcus aureus
(Kluytmans et al, 1997). Stapylococcus epidermidis detected on the surface of the soft toy is
also found as part of our skin flora and therefore it is understood to be, for the most part,
harmless. There are exceptional circumstances however such as in humans that are
immuno-compromised (Salyers and Whitt, 2002). Furthermore, due to various methods of
contamination, it is known to be probably the most common microorganism species found
when performing tests within a laboratory (Queck and Otto, 2008).
~ 26 ~
Mycobacterium phlei was the unknown identified last during the research undertaken.
Being Mycobacterium it was initially difficult to classify as simple staining, such as that of the
gram stain, does not provide good, conclusive results as to whether this particular unknown
was indeed gram positive or negative. The reason because of this is due to the presence of
the long chain fatty acid, mycolic acid, within the cells walls of bacterium of the particular
the genus (Vance et al, 1973). Mycobacterium phlei, much like Bacillus subtilis is also found
principally on grass and more importantly is known to be non-pathogenic in humans.
From the results gathered and obtained from laboratory experimentation it is evident that
amongst almost all the unknowns identified there is no immediate threat to the children of
the child-care environment. Bacillus cereus poses the most cause for concern as it is a well
defined pathogen in humans causing only a small minority of all food-borne
illnesses (Kotiranta et al, 2000). However, it should also be understood that the bacteria that
were found to be present on the surface of the toys are ever-present whether they remain a
part of our external environment or even our internal environment making up much of our
normal human flora.
~ 27
28 ~