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Table of Contents
ABSTRACT .................................................................................................................................................. 1 1.0 INTRODUCTION .................................................................................................................................. 2 1.1 Introduction ................................................................................................................................ 2 1.2 Objectives .................................................................................................................................. 3 2.0 METHODOLOGY ................................................................................................................................. 3 3.0 RESULTS AND DISCUSSIONS………………………………………………………………………5 4.0 CONCLUSIONS…………………………………………………………………………..……..……11
5.0 REFERENCES………………………………………………………………………………………...12 6.0 APPENDICES………………………………………………………………………………………...13
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ABSTRACT
Mould and yeast usually make the food become hazards through their excretion of toxins and spore although some of yeast may have advantages in food industry in certain amount. There are differences that can be determined between mould and yeast although both of them are under the classification of fungi. The differences are observed on the cellular morphology and colony morphology. Moulds are seen to have larger colonies than yeast. From the cellular morphology, the presence of hyphae and structure of moulds completely different from yeast which is a unicellular microorganism. Yeast and moulds are compared through the fruit juice (mango juice). Besides, from the enumeration of these microbes on PDA, YMA and DRBCA, shows that the most suitable agar which support their growth especially for yeast was the Potato Dextrose Agar (PDA). DRBCA seems to contain substance that restrict the spread of mould, thus make it smaller compared to the mould that growth on PDA and YMA.
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1.0 INTRODUCTION 1.1 Introduction
Fungi consist of yeasts and moulds which have been classified to sub-nature based on mycelia septation, and phyla according to its ability to produce spores and types of sexual spore produced. Both yeasts and moulds spoil the food, thus, it is important to control the sanitary and safety the foods although it had been cooked. Some fungi may secrete toxin which is mycotoxin that hardly damaged even in the extreme condition. Yeasts are unicellular fungi while moulds are larger and easily detected on the food. This is because moulds grow as a filament (mycelia) structure. Saccharomyce cervisiae is a type of yeast that can be used in the baking process. Although yeasts and moulds may hazardous to human body, but still there are some advantages that can be obtained from them. There are several ways to enumerate and detect the yeasts and moulds. Several media are used such as acidified Potato Dextrose Agar (PDA), Dichloran Rose Bengal Chloramphenicol Agar (DRBCA), and Yeast and Mould Agar (YMA). Other than Saccharomyce cervisiae, other moulds which are Aspergillus niger and Aspergillus braseliensis
also can be enumerated on those agar. Potato Dextrose Agar (PDA) is a nonselective medium for the enumeration of yeasts and moulds. It is used as general medium for yeasts and moulds and can support their growth as PDA is composed of dehydrated Potato Infusion and Dextrose. Potato infusion may encourage mould sporulation and production of pigments in some dermatophytes. Besides, the bacterial is inhibited from grows through the addition of tartaric acid to lower the pH of PDA to 3.5. Another media used is the Dichloran Rose Bengal Chloramphenicol Agar (DRBCA). This media will inhibit the spreading of moulds and restrict the colony size as there is the presence of Dichloran. Besides, Chloramphenicol is used to inhibit the growth of gram-negative bacteria. Enumeration of yeast and mould are essential with the Dichloran Medium Base with Rose Bengal (DRCB) while peptic digest of animal tissue provides the carbon and nitrogen for the growth. Yeast and Mould Agar also used in this experiment for the enumeration of yeast and moulds.
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1.2 Objectives
From this experiment, the quantity of yeast and moulds can be identified and compared in fruit juices. Besides, the media used to enumerate yeasts and moulds are able to be compared. In addition, macroscopic and microscopic appearance of food-borne yeasts and moulds also able to be described. 2.0 METHODOLOGY Materials and Equipments
Commercial juice or juice extracted in the lab
Pure culture of Saccharomyces cerevisiac yeast and Aspergillus niger mould
Two 9 ml MRD diluents
6 petri dishes containing acidified PDA ( Potato Dextrose Agar )
6 petri dishes containing DRBCA ( Dichloran Rose Bengal Chloramphenicol Agar )
6 petri containing YMA ( Yeast and Mould Agar )
Alcohol
Spreading glass
Micropipette ( 1000 µl ) and pipette tip
Incubator, set at 25 C
o
Methods
A) Sampling and dilution preparation 1. 1 mL of mango juice is transferred aseptically into 10ml of MRD diluents. -1
-2
2. 10 and 10 dilution is prepared by using 9ml of MRD diluents. B) Acidified PDA, DRBCA and YMA plating 0
1. 0.1mL of 10 dilutions is divided into plates consists acidified PDA, DRBCA and YMA (each plate is triplicated). -1
-2
2. Same step is repeated to 10 and 10 dilution. 4
3. The amount of plates will be nine types of media. 4. Inoculated juice sample is spreaded into plate by using sterilized gas spreader. o
5. Plates are incubated upside down, at 22 C for 7 days. 6. Steps 1-3 is done for pure culture for prepared dilution. C) Colony counting and morphology inspection Materials and Equipments
Incubated petri dish (acidified PDA, DRBCA and YMA)
Lactophenol cotton blue colourant
Transparent adhesive tape
Crystal violet
Microscope
Colony observation
Yeast 1. 3 yeast colonies represent different colony morphology is picked from those plates. 2. Some colonies are transferred on microscope slide that contain one drop of water. 3.
A smear is made on the slide.
4. Crystal violet is applied as simple colorants onto the smear for about 60 seconds. 5.
Yeast cells are checked using oil ommersion microscope (100X).
6. Cellular morphology for yeast is drawn.
Mould 1. One drop of Lactophenol cotton blue is placed on the glass slide. 2. The surface of mould colony is slowly touched using adhesive tape. 3. Adhesive tape is put on the colorants. 4. The slide is checked using microscope. 5. Cellular morphology for mould is drawn. 6. If coloring is succeeded, mycelia structure and reproduction will visible. 5
7.
Some slides are prepared to show how hard it is to press and transfer mould to adhesive tape correctly.
8. Those steps are repeated to the three mould colonies, with different morphology are able to be checked. 9. Observations are recorded.
3.0 RESULTS AND DISCUSSIONS
In this laboratory session, variety of fruit juice is tested to identify and compare the quantity of yeast and moulds. Each group was given different fruit juice. Fruit juices that had been tested were mango juice, kiwi juice and dragon fruit juice. The positive control used was Saccharomyces cerevisiac for yeast while Aspergillus niger and Aspergillus brasiliensis as
positive control for moulds. Serial dilution was prepared before the enumeration to obtained a 0
countable colonies and avoid them from clumping. The serial dilution was prepared for 10 , 10 -2
-1
0
and 10 in which the 10 is a pure juice. The serial dilution was prepared by using the Maximum Recovery Diluent (MRD). This diluent is an isotonic substance to ensure the recovery of organisms from various sources. Besides, it combines the protective effect of peptone with the osmotic support of physiological saline. Spreading of 0.1 ml of dilutions sample in different media shows different results in the amount of colonies formed. Different growth media was used to see the differences. Media that had been used was acidified PDA, DRBCA and YMA.
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Growth
Replication
Media
Sample
Colonies Amount
Volume
10
10
10
(cfu/ml)
TNTC
TNTC
153
164500
0
Amount
-1
-2
(ml) Acidified
1
PDA
2
TNTC
TNTC
TNTC
3
TNTC
TNTC
176
TNTC
TNTC
114
2
TNTC
TNTC
136
3
TNTC
TNTC
83
TNTC
TNTC
TNTC
2
TNTC
TNTC
156
3
TNTC
TNTC
TNTC
DRBCA
YMA
1
1
0.1
0.1
0.1
111000
156000
Table 1: Growth of colonies on media Table 1 show that the highest amount of colony forming unit was obtained on the acidified PDA, followed by Yeast and Mould Agar and DRBCA. Most of the colonies are more than 200 and 0
-1
stated as too numerous to count (TNTC) in the sample dilution 10 and 10 . The results recorded in the table above are obtained from the mango juice. Potato dextrose agar is a media rich in dehydrated potato infusion and dextrose which will encourage the growth of yeasts. Yeasts will ferment the sugars present and produced carbon dioxide and ethanol. Those sugars present in the potato and dextrose will give more energy for the growth of yeasts. Yeasts that grew on the acidified PDA were observed to have brown colonies. As acidified PDA is not a differential agar for yeasts, hence, the amount and colonies that appear cannot be identified either it was the Saccharomyces cerevisiac or not without observing under the microscope. Acidified PDA will inhibit the growth of bacteria as reported by F. Eugene Nelson on 1972. On the other hand, the growth of Aspergillus niger on acidified PDA was observed as black moulds and there are too numerous. It appears as bigger colonies compared to the yeasts. The colonies grow as conidial and the radiate can be seen. In Yeast and Mould Agar, the colony forming units was slightly lower than acidified PDA. Colonies was observed to have light brown in colour. YMA consists of yeast extract, malt extract, peptone, dextrose and agar. The sugar contains in YMA is lower than acidified PDA. 7
The YMA is recommended for the isolation and maintanence of yeasts and moulds. In addition, it is used for detection of both Saccharomyces and non- Saccharomyces yeast. Wild yeast might grow in the agar plate. As in the acidified PDA, acid is presence in the agar, however, the pH of YMA is slightly higher than the pH in acidified PDA which is 4.0 compared to 3.5 in acidified PDA. Hence, with the lower contents of sugar for fermentation, and slightly higher pH, the colonies formed on YMA were lower compared to acidified PDA. The lowest colony formed was on the DRBCA. DRBCA was composed of glucose, bacteriological peptone, potassium phosphate, magnesium sulfate heptahydrate, Rose Bengal dichloran solution, chloramphenicol, distilled water and agar. The content of glucose was lowest in DRBCA compared to PDA and YMA. Hence, this will restrict the fermentation and enumeration of the yeasts on DRBCA. Besides, the addition of dichloran and rose Bengal effectively slow down the growth of fast-growing fungi. Hence, the detection of other yeasts and mould can be done on those microbes that have lower growth rates. Moreover, the pH of DRBCA was the highest which is 5.6 which will slower the growth rates of yeasts. Colonies that had been observed on the DRBCA was pink, dark pink and white in colour. However, there is growth of mould which being observed to have white furry surface on one of the sample dilution. Pink colonies was appear from the effect of rose Bengal, when the yeast had absorbed the rose Bengal and make it easier to be seen and counted. After calculate the colony forming unit, cellular morphology was observed under the microscope from food sample to compare with the positive control. The colony morphology is different within the media. The difference are clearly seen through the colour of colonies grow from yeast. The difference and description of colony morphology and cellular morphology are described as below;
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Sample (Juice)
Colony Morphology
Cellular Morphology
Yeast
PDA; Round colonies, brown in colour
Coccus
YMA; Round colonies, light brown in colour
grape-like colonies. Presence
shape,
chain
and
DRBCA; Round colonies, pink, light pink of red colour within the blue
Mould
and white in colour.
coccus cell. Unicellular.
Filamentous form, filiform margin, black.
Presence of hyphae, colour,
fruiting
black
structure,
radiate structure. Presence of small
coccus
cell
(red)
surrounding. Table 2: Colony and Cellular Morphology of Yeast and Mould Colony morphology was observed through the yeasts and moulds appearance on the plate agar while the cellular morphology was observed through simple staining under the microscope. There are difference way of staining between the yeast and moulds. Staining for yeast only by using the crystal violet as simple colorants to give colour to the cell and make it clear and easily to be seen. Yeast was observed to have grown in a unicellular and grows in chain or group. On the other hand, moulds which is Aspergillus niger and Aspergillus braseliansis are stained with Lactophenol cotton blue. Lactophenol cotton blue was used to make the observation under the microscope become easier. Both moulds show the appearance of the hyphae or conidiophores. The moulds were seen to have black sterigmata. However, the sterigmata and vesicles were not able to differentiate on the microscopic observation. The colony morphology shows that the mould is in filamentous form, have filiform margin and black in colour. However, the moulds that grow on the DRBCA were smaller and less spread. This is the result from the presence of dichloran in that media.
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Moulds and yeast sometimes give advantages in the food industry through their ability to ferment glucose and being use in the production of wine and bread. However, if the yeast and moulds are over than required, it may be harmful as they have the ability to produce toxin.
QUESTIONS AND ANSWER
1. The mode of action of DRBCA is that the medium contains Dichloran and rose bengal are added to inhibit the invasion of fast-spreading moulds and reduces the size of other genera colonies, improving the colony count. Rose bengal is also easily absorbed by yeasts which tend to dye them to pink. Besides that, rose bengal is also acts as a selective agent that inhibits the growth of bacteria, limits the size and height of faster-growing moulds, allowing for the development and detection of other slower-growing yeasts by dyeing them to pink. Chloramphenicol acts as the selective agents that inhibit the growth of bacteria. It is a recommended antibiotic for neutral media like DRBCA due to its heat stability and wide bacterial spectrum. The mode of action of YMA is that the medium contains lactic acid that reduces the pH of the medium to 4.0 to render the selectivity of the medium. YMA has been formulated for the suitability for the detection of both Sachharomyces and non Saccharomyces wild yeasts in the presence of yeast culture.
2. Moulds can be harmful; but in other ways, they can be beneficial. Moulds are different kinds of fungi that thrive on other living things by the mean of either sexual or asexual sporulation. In general, human classified moulds as organisms that imply rotting and cause food spoilage. However, contrary to popular beliefs, moulds can be very beneficial too. Here is a list of some of its benefits: a) Moulds can be used in the cheese production to give the product a distinct flavor. For example, the blue cheese is made by using Penicillium glaucium which give the cheese a distinctive blue color. Moulds are also known for its benefits in the production of tempeh, Puer tea and Quorn. Other than this, the moulds can be also eaten in the form of mushroom because mushroom is one of the moulds that can be eaten. b) Moulds can be used in the manufacture of antibiotics. This is based on the facts that moulds are known for its inherent protections against certain kinds of 10
bacteria. For example, penicillin is made by using P. notatum which is widely accepeted in the medical industry as the best drug to destroy or decelerate the growth of bacteria. c) Moulds are also used in the fermentation of organic acids. This is because moulds are able to accelerate the fermentation of the organic acids to retain more nutrients needed in the production of organic acids. Through fermentation, moulds are also used to make beer and breads. d) Moulds are used to accelerate the decomposition of a dead substance to produce free carbon for the maintenance of life on the Earth. Without moulds, decomposition will not take place; the carbon atoms will not be reprocessed. This will affect the production of carbon in the environment. If this happen, the life on Earth are not able to survive. The moulds are known to produce mycotoxin which is able to bring many harmful effects to the human body. Here is the list of harmful aspects of the moulds. Long-term exposure to the moulds is unhealthy to anyone, especially the infants and children, elderly people, individuals
with
respiratory
conditions,
allergy
and/or
asthma
and
those
immunocompromised patients. Generally, the exposure to the moulds is associated with the allergic symptoms; for example, nasal and sinus congestion, sore throat, cough, dyspnea, asthma, epistaxis, upper respiratory tract infections, headache, skin and eye irritation. Those mentioned above is some of the mild cases. However, some moulds are capable of producing extremely potent toxins, mycotoxins that are lipid-soluble and readily absorbed by the intestinal lining, airways and skin. These agents usually contained in the fungal spores. The mycotoxins are able to cause severe diseases. Some of them are cancer (alfatoxin bst characterized as human carcinogen), hypersentivity pneumonitis/pulmonary fibrosis, pulmonary injury, neurotoxicity, hematological and immunological disorders, hepatic, endocrine and/or renal toxicities. 3. Yeast is classified as fungus, a eukaryotic microorganism. It is able to reproduce sexually and asexually. Yeast reproduces sexually with the production of ascospore in an ascus as the fungi do. Thus, they are the members of the Ascomycota in the Kingdom Fungi. Other than this, yeasts are also heterotrophic as all the fungi. Yeasts are the same as other fungi that act as the saprophytes that decay dead materials. Other than this, yeast is also 11
able to reproduce asexually as the other fungi do. As the fungi, yeasts also play important role in the fermentation of certain food products to increase the quality of the foods. Yeast is differed with other fungi because it is a unicellular organism; while the others are multicellular organisms. Other than this, unlike other fungi, yeasts reproduce by the mean of budding or binary fission while others use sporulation. The symmetrically division of the cells is known as binary fission; while the cleavage of yeast cells which are asymmetrical is considered as budding. Besides, yeast does not have hyphae to hold the surface; yeasts also do not have pin-like projections of sporangia to disperse the spore to surrounding because yeasts reproduce by budding. As compared to moulds, yeasts grow more slowly; normally take a week of time to grow. 4. Buds are the daughter yeast cells attach to the parent yeast cells to obtain nutrients for maturation process. Buds are produced by the asymmetrically cleavage of the yeast cells which are smaller than the parent cells. The buds will then grow to form a mature cell and detach from the parent cells to form individual cell. Ascus is the sexual spore-bearing cell produced in ascomycete fungi. An ascus normally contains eight ascospores, produced by meiotic division followed by a mitotic division after the conjugation with other organisms under drastic condition. An ascospore is a spore contained and produced in an ascus. Typically, a single ascus will contain 8 ascospores. This is the products of sexual reproduction of the ascomycete fungi. The sporulations enable the organisms to survive under drastic condition because the spore is heat resistance and which will germinate during suitable condition.
4.0 CONCLUSIONS
Fruit juice also has the presence of yeasts and moulds. Yeasts and moulds will produce toxin that will be harmful towards the body system. Potato Dextrose Agar was the best agar to enumerate and supply the needed of yeasts and moulds for growth. Further, detailed in staining and enumeration on different type of yeasts or moulds can be observed to detect the type of yeast present in the fruit juice. Besides, more type of media can be used to enumerate the yeasts and moulds.
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5.0 REFERENCES
Data
sheet:
Potato
Dextrose
Agar
(2011)
downloaded
from
http://www.neogen.com/acumedia/pdf/ProdInfo/7149_PI.pdf on 26th October 2011. Data
sheet:
DRBC
Agar
7591,(2011)
downloaded
from
http://www.neogen.com/Acumedia/pdf/ProdInfo/7591_PI.pdf on 26th October 2011. Data
sheet:
Yeast
and
Mould
Agar,
(2011)
downloaded
from
http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0920&org=149&c=UK&lan th
g=EN on 26 October 2011. Data
sheet:
Rose
Bengal
Chloramphenicol
Agar
(2011)
downloaded
from
http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0549&org=149&c=UK&lan th
g=EN on 26 October 2011 Internet:
Shijun
Liu
(2011).
Interpreting
Plates.
Downloaded
from
http://www.sciencebuddies.org/science-fairprojects/project_ideas/MicroBio_Interpreting_Plates.shtml on 27th October 2011 Internet: Mike Morgan (1999). Moulds. Downloaded from http://www.microscopyth
uk.org.uk on 26 October 2011 Koneman, E.W., et. al. (1997). Color Atlas and Textbook of Diagnostic Microbiology .5th Ed. Philadelphia: Lippincott Murray, P.R., et. al. (1995). Manual of Clinical Microbiology. Washington, DC: American Society for Microbiology.
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6.0 APPENDICES
Figure 1: Mango juice (sample)
Figure 2: Dropping of sample dilution on plate agar
Figure 3: Spreading of sample dilution on plate agar
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Figure 4: Transferring moulds (pure) into DRBCA
Figure 5: Mould ( Aspergillus niger )
Figure 6: Yeast ( Seccharomyces cerevisiae)
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Figure 7: Growth of Saccharomyces cerevisiae (left) and Aspergillus niger (right) on PDA
Figure 8: Growth of Aspergillus niger (left) and Saccharomyces cerevisiae (right) on YMA
Figure 9: Growth of Aspergillus niger (left) and Saccharomyces cerevisiae (right) on DRBCA
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Figure 10: Growth of yeast and moulds from sample on PDA
Figure 11: Growth of yeast and moulds from sample on YMA
Figure 12: Growth of yeasts and moulds from sample on DRBCA
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Figure 13: Saccharomyces cerevisiae observed under 100× of magnification
Figure 14: Aspergillus niger observed under 100× of magnification
Figure 15: Aspergillus brasiliensis observed under 100× of magnification
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Figure 16: Yeast colony from sample observed under 100× of magnification
Figure 17: Mould colony from sample observed under 100× of magnification
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