the amount of microorganisms in the oral fluid ( saliva ) ranges from 4 millions to 5 billions per 1 milliliter , and in dental plaque between 10 and 1000 billions per 1 gram . their resistance to non - specific oral protective factors and environmental factors ( e.g. , antiseptic solutions ) depends on the cellular membrane , a thick layer of mucus , a capsule , or other biological characteristics . for this reason , standard bacterial cultures with differing biological characteristics were selected for this study : staphylococcus aureus are gram - positive cocci found in the nostrils of up to 60% of humans ; they form microcapsules , and are resistant to high temperatures ( up to 70c ) , disinfectant , and antiseptic solutions , and cause suppurative inflammatory processes . staphylococcus aureus is an important pathogen because of its virulence , antimicrobial resistance , and association with various diseases , including fatal systemic infections , intoxication , cutaneous infectious , and opportunistic diseases . klebsiella pneumoniae are capsule - forming bacteria found in the respiratory tract and are capable of causing atypical pneumonia . candida albicans is a fungus and is part of the normal microflora of the oral cavity , but it can cause an opportunistic infection in immuno - depressed subjects . escherichia coli is part of the normal microflora of the large intestine ; however , due to poor hygiene it can enter the oral cavity and cause suppurative inflammatory processes . the procedures that a prosthodontist performs may cause damage to the patient s oral mucosa and gingiva . the saliva mixes with blood . during the manufacturing of the dental impressions , blood and saliva enter the setting impression material , and it becomes contaminated with bacteria and viruses [ 47 ] . for this reason , according to general hygiene norms , such impressions should be disinfected ; yet disinfection does not always yield the desirable results . when producing gypsum casts using non - disinfected or incompletely disinfected dental impressions , the microorganisms travel from the surface of the impression material into the cast . the infected material then is taken to the manufacturing facilities and may pose threat to dental technicians . dental casts are subject to several forms of contamination , ranging from direct contact with a patient s saliva , to procedures involving measurement , planning , manufacture , laboratory shipping , and storage . dental technicians touch the impressions or the casts with their hands , and their skin is frequently damaged due to their work . when cutting the dental casts , bacteria , fungi , or particles of the disinfectant may travel with the plaster dust from the material to the respiratory tract of personnel , causing infections , and may also accumulate on the clothing or open surfaces in the room . the aim of study was to evaluate the survival of staphylococcus aureus , klebsiella pneumoniae , and escherichia coli bacteria , and candida albicans fungus in non - disinfected dental casts . the results of the study could be used to determine how long dental casts may pose threat to the health of the personnel and the patients . perhaps disinfection of dental impressions would be sufficient , skipping additional disinfection of the dental casts in order to reduce exposure of the personnel to possible allergens  the residues of disinfectant solutions in the plaster . although extensive research has been carried out , there are no published studies in which similar issues were addressed . during this experimental study we investigated the survival of the selected bacteria and fungi in dental casts . the study was conducted using standard bacterial ( staphylococcus aureus atcc 25928 , klebsiella pneumoniae atcc 13883 , and escherichia coli atcc 25922 ) and fungal ( candida albicans atcc 60193 ) cultures . saline suspensions ( mcfarland turbidity standard 0.5 ) were prepared using standard bacterial cultures cultivated in trypticase soy agar for 24 hours at 3537c , and candida albicans culture cultivated in sabouraud agar for 3 days . we evaluated and compared changes in the number of the microorganisms in gypsum casts at different time intervals . gypsum casts ( an equivalent to dental cast ) were manufactured in a sterile laminar - flow hood under sterile conditions , using standard alabaster plaster class 2  moldabaster the plaster was sterilized ; therefore , there was no need to form a control group . the dental casts were prepared by pouring 65 g sterile plaster powder , 5 ml sterile distilled water , and 1 ml of a specific bacterial or fungal suspension ( mcfarland turbidity standard 0.5 ) into a sterile mixing container . a sterile spatula was used to stir the contents until an integral mass was formed , which was subsequently placed into a sterile closed container . during the test period the closed containers with the samples were placed on a dental lab shelf to simulate casual cast storage conditions . the number of the aforementioned standard microorganisms per 1 g of the substance was evaluated after 1 , 2 , 24 , 48 , 72 , 96 , and 120 hours . sterile clamps were used in the making of each sample . the sample  a piece of the gypsum cast of a known weight ( ca . 1.0 g )  was ground to powder in a sterile mortar , and then placed into a sterile glass with 5 ml of sterile saline solution . the suspension of plaster powder was diluted at the ratio of 1:5 , 1:25 , 1:125 , and 1:625 . one ml of the suspension from each test tube with different dilution ratios was poured into 10 ml of 45c trypticase soy agar ( or sabouraud agar , in the case of candida albicans ) in 2 petri dishes that were then stored at 3537c ( in case of candida albicans , sabouraud agar was stored at 2025c ) . after the cultivation of the indicated duration , colonies of the respective bacterium or fungus in the 2 petri dishes were evaluated , calculating the mean number of the colonies ; multiplying this number by the degree of dilution revealed the number of bacteria or fungus per 1 g of plaster . the data were considered to be statistically significant if the probability of an error was p<0.05 . the study showed that k. pneumoniae survived the longest in gypsum casts  up to 4 days  compared to the other studied microorganisms ( figure 1 ) . a statistically significant reduction in the number of k. pneumoniae ( from 837911935 to 44120845 ) in gypsum casts was observed after 1 day ( p<0.05 ) . the number of k. pneumoniae colonies was also the highest , compared to other bacteria . after the first and the second hour in the gypsum cast , the number of bacteria remained nearly unchanged . it was only after 2 days that a statistically significant difference was detected ( a reduction from 59154676 to 9285102 , p<0.05 ) . gram - positive bacteria ( staphylococci ) remained viable for up to 3 days in the gypsum casts , presumably due to their cell membrane composed of a thick peptidoglycan layer , a microcapsule ( a thin layer of mucus over the cell membrane ) , and high resistance to environmental factors ( figure 2 ) . no gram - negative bacteria ( e.g. , e. coli ) were found in gypsum casts after 2 days , and a statistically significant reduction in their numbers was observed just after 2 hours ( from 19483336 to 470574 , p<0.05 ) ( figure 3 ) . microorganisms with a eukaryote structure ( e.g. , the fungus c. albicans ) died within 2 days , and a statistically significant reduction was observed after 1 day ( from 31 3662759 to 12 2911234 , p<0.05 ) ( figure 4 ) . figure 5 presents the comparison of the standard microorganisms amounts ( the bacteria and the fungi ) at different time intervals , and the quantitative reduction in their numbers . the quantitative evaluation of the viability of standard microorganisms in gypsum casts showed that physical - chemical characteristics of gypsum casts have different effects on the survival of the standard microorganisms . structural and physiological characteristics of k. pneumoniae and s. aureus affect their survival ( their quantity ) with respect to that of e. coli and c. albicans . the findings of the study showed that the microorganisms did not multiply in the gypsum casts ; instead , their numbers significantly dropped . the study was conducted using the most common oral bacteria and fungus : staphylococcus aureus , klebsiella pneumoniae , candida albicans , and escherichia coli . studies performed in japan showed that all the microorganisms used in our study  staphylococcus aureus , klebsiella pneumoniae , and escherichia coli  were detected in the oral cavities of 13.6% of patients with no health complaints . according to other authors , staphylococcus aureus is detected in the oral cavities of 15.8% of patients , and candida albicans in 63.0% of patients . the findings of our study showed that the duration of the survival of microorganisms in gypsum casts varied depending on the types of the studied cultures . these bacteria have a thick layer of peptidoglycan and a microcapsule , which is a mucous layer of polysaccharide origin . the gram - negative bacteria klebsiella pneumoniae and escherichia coli differ from staphylococci in their cell membrane structure . however , klebsiella bacteria have a thick mucous layer of polysaccharide origin  a capsule  that ensured their longest survival in gypsum casts compared to other microorganisms . escherichia coli have no capsule and thus are more susceptible to unfavorable environmental conditions , thus these bacteria survived in gypsum casts for only 12 days . the fungus candida albicans , although it has a eukaryote cell structure , survived in gypsum casts longer than escherichia coli did because fungi have a chitin membrane . the oral cavity may contain pathogenic as well as relatively pathogenic microorganisms that can be transferred into the environment through impressions . in patients with damaged oral mucosa , staphylococcus aureus , klebsiella pneumoniae , and escherichia coli may cause suppurative inflammatory processes . escherichia coli may cause not only a local inflammatory process , but bacteremia as well . candida albicans , as part of the normal oral microflora , is found in 4060% of individuals ; however , this fungus can cause candidiasis in immuno - compromised states or in cases of antibiotic overuse . most commonly , alginate and silicone dental impressions are used in clinical practice . due to their composition , structure , and the hydrophilic setting mechanism , alginate impressions are easily infected by oral microorganisms . a more monolithic structure of the silicone impression mass and its non - hydrophilic setting mechanism significantly reduce the attachment of microorganisms to the impression surface . selecting a suitable impression mass may reduce the spreading of the infective agents as early as the first stages of prosthesis manufacturing . alginate impressions are more frequently contaminated with oral bacteria and fungi , compared to silicone impressions , and they are also more difficult to disinfect . alginate impressions pose a significantly higher threat of contaminating dental casts with bacteria and fungi , compared to silicone impressions , thus silicone impressions are safer with respect to the transmission of the infective agents . previous studies have shown that dental impressions delivered at dental laboratories contain bacteria irrespectively of whether they were disinfected or washed under running water . this suggests that dental casts produced using well - disinfected impressions require no additional disinfection . if the dental casts are stored under normal conditions of a dental technician s office , the number of microorganisms should change more rapidly than it did during our study due to frequently changing temperature and humidity , and because of the plaster ph of 6.1 . an additional disinfection of dental casts would not yield significant results , compared to the risk that the dental technicians would face because of exposure to a potential allergen  the disinfectant on the surface of the dental cast . although studies have shown that the effect of the disinfectant on the precision of the dental casts is not significant , the risk remains that the disinfectant may alter the physical characteristics of the dental cast surface , making it more porous , brittle , and more susceptible to wear and tear . rinsing of the oral cavity with an antiseptic solution prior to odontological procedures may reduce the presence of microorganisms in the oral cavity . the most popular oral antiseptic , chlorhexidine , is not always effective against fungi or bacterial spores . thus , mouth rinsing with an oral antiseptic prior to taking impressions followed by full disinfection of the impressions would reliably reduce the contamination of dental casts with microorganisms . as our results show if the impression is disinfected and pre - impression mouth rinsing is used consistently , the dental casts would pose less threat because of the poor survivability of bacteria and fungi in gypsum . gram - positive bacteria staphylococcus aureus and gram - negative bacteria klebsiella pneumoniae demonstrated the longest survival in dental casts ( up to 4 days ) , compared to escherichia coli and candida albicans , which remained viable during the first 2 days ) . dental casts are not a suitable medium for the multiplication of the colonizing microorganisms . individual protective measures ( gloves , protective goggles , and respiratory protection measures ) should be used in order to avoid possible contamination of dental impressions and casts with microorganisms , and to prevent their transmission and spread in environments such as dental laboratories .