initiative 
Harald Löe, Else Theilade and S. Börglum Jensen. Experimental Gingivitis in Man. Journal of Periodontology May-June 1965, Vol. 36, No. 3: 177-187.
The objective of this study was to observe clinical and microbiological changes in a group of patients who completely discontinued their daily oral hygiene routines.
Twelve (12) subjects were chosen from the School of Dentistry. At the start of the study, each participant was given a clinical examination to determine gingival index (GI) and plaque index (PI). Each of these indices were recorded for three parts of the teeth: buccal, lingual and proximal, and individual tooth scores were grouped to calculate an average per tooth group: incisors, premolars and molars.
Once initial registrations were taken, patients stopped using any form of oral hygiene, and during this time, registrations continued to be taken. As soon as gingival inflammatory changes were clear, between 2 and 3 weeks, clinical indices were again measured, and subjects were asked to brush their teeth and to use wood sticks to massage their gums interdentally. When gingival and plaque indices returned to their baseline values, the experiment was concluded. Bacteriological assessments were also performed throughout the course of the study using impression and bacterial smear preparations.
Levels of oral hygiene and gingival conditions were quite good for all individuals at the start of the trial, and over the course of the study both steadily worsened. No differences were observed between the upper and lower jaws. There was a tendency for greater inflammation in the interproximal surfaces. The areas with the least amount of plaque and inflammation overall were the lingual surfaces and particularly the upper premolars. Once oral hygiene routines resumed, both IP and GI dropped dramatically, to even lower than baseline.
Bacteriological examination was performed in three different phases. At the beginning of the non-brushing period, cocci greatly increased in quantity, and masses of desquamated epithelial cells and some leukocytes were observed. A few days later, a predominance of filamentous forms and slender rods were observed, along with quite large amounts of cocci and a great increase in leukocytes. In the last phase, between 6 and 10 days, depending on the subject, great amounts of vibrios and spirochetes along with large quantities of leukocytes and some cocci could be detected. When oral hygiene techniques resumed and gingivitis disappeared, samples were dominated by cocci with some short rods, but in no case were vibrios or spirochetes found.
This study clearly showed the absence of oral hygiene to cause a dramatic increase in plaque accumulation and in gingival inflammation in all of the subjects and in all of the areas of the mouth within a period of 2-3 weeks. The lingual surfaces accumulated the least amounts of plaque and experienced the least inflammation, indicative of the tongue’s self-cleansing ability. With regard to the bacteriological examination, healthy gums were proven to hold few bacteria, these being predominantly cocci, and that plaque maturation produces changes in the environment that allow new species to grow.
Therefore, it is clear that the accumulation of bacterial plaque was the cause of gingival inflammation, although the exact underlying causal mechanisms were left unknown. Plaque maturation and the fact that the late colonising species emerged before signs of clinical gingivitis, indicated that these species had something to do with the development of the inflammation. Variations in the appearance of the gingivitis explain the variability that exists in the defense mechanisms of the different individuals.
Lastly, within days after reinitiating oral hygiene methods, all subjects were observed to restore the healthy state of their gums, regardless of how long it took them to clinically develop the gingivitis.